DE69937419T2 - Neisseria meningitidis antigens and compilations - Google Patents

Abstract

The invention provides proteins from Neisseria meningitidis, including the amino acid sequences and the corresponding nucleotide sequences. The proteins are predicted to be useful antigens for vaccines and/or diagnostics.

Description

FIELD OF THE INVENTION

These The invention relates to antigens of the bacterial species: Neisseria meningitidis and Neisseria gonorrhoeae.

BACKGROUND

Neisseria meningitidis is a non-motile Gram-negative diplococcus pathogen in humans. It colonizes the pharynx, causing meningitis and occasionally septicemia without meningitis. It is closely related to N. gonorrhoeae, though a feature that clearly distinguishes Meningococcus from Gonococcus, the presence of a polysaccharide capsule is common to all pathogenic meningococci.

N. meningitidis causes both endemic and epidemic disease. In the United States is the infestation rate 0.6-1 per 100,000 people per year, and it can be essential during the outbreaks be higher (See Lieberman et al., (1996) Safety and Immunogenicity of a Serogroups A / C Neisseria meningitidis Oligosaccharide Protein Conjugate Vaccine in Young Children. JAMA 275 (19): 1499-1503; Schuchat et al., (1997) Bacterial Meningitis in the United States in 1995. N. Engl. J. Med. 337 (14): 970-976). In developing countries Endemic disease rates are much higher, and while Epidemics can reduce the frequency 500 cases reach per 100,000 people per year. Mortality is extreme high, 10-20% in the United States and much higher in developing countries. To introduction of the conjugate vaccine against Haemophilus influenzae is N. meningitidis in the United States the largest causative agent of bacterial Meningitis at all ages (Schuchat et al., (1997), supra).

Based on the capsular polysaccharide of the organism are 12 serogroups of N. meningitidis. Group A is the pathogen the most common on the epidemic disease in the sub-Saharan African region is involved. Serogroups B and C are in the vast majority of cases United States and responsible in most developed countries. For the remaining cases in the United States and developed countries The serogroups W135 and Y are responsible. The currently used Meningococcal vaccine is a tetravalent polysaccharide vaccine, which is composed of serogroups A, C, Y and W135. Although he is effective in adolescents and adults, he calls only one low immune response and a short protection period and can not be used in children [z. Morbidity and mortality weekly report, vol. 46, no. RR-5 (1997)]. This is due to the fact attributed to that Polysaccharides are T cell independent antigens which cause a weak immune response by repeated immunization not reinforced can be. After the success of the vaccine against H. influenzae are Conjugate vaccines against serogroups A and C have been developed and are in the final stage of clinical trials (Zollinger WD "New and Improved Vaccines Against Meningococcal Disease. "In: New Generation Vaccines, supra, pp. 469-488; Lieberman et al., (1996), supra; Costantino et al., (1992) Development and Phase I clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10: 691-698).

meningococcus B (menB), however, remains a problem. This serotype is currently for about 50% of all meningitis disorders in the United States, Europe and South America responsible. The polysaccharide approach can not be used because the capsule's polysaccharide polysaccharide is a polymer of α (2-8) -linked N-acetylneuraminic acid, which also in the tissues of mammals occurs. This results in tolerance to the antigen; indeed would one caused immune response to be directed against itself and therefore undesirable. To avoid causing an autoimmunity and a protective immune response For example, the polysaccharide of the capsule is chemical been changed, by replacing the N-acetyl groups by N-propionyl groups which leave the specific antigenicity unchanged (Romero & Outschoom (1994) Current Status of Meningococcal group B vaccine candidates: capsular or noncapsular? Clin. Microbiol. Rev. 7 (4): 559-575).

Alternative approaches to menB vaccines have used complex mixtures of outer membrane proteins (OMPs) that either contained the OMPs alone or poragen-enriched OMPs or deleted the class 4 OMPs that are believed to be antibodies induce bactericidal activity. This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but by and large are not effective when there are numerous antigenic variants of the outer membrane proteins. To the antigenic To overcome variability, multivalent vaccines containing up to nine different porins have been developed (eg, Poolman JT, (1992) Development of a Meningococcal Vaccine., Infect. Agents Dis., 4: 13-28). Additional proteins that have been used in the outer membrane vaccines have been the opa and opc proteins, but none of these approaches has been able to overcome antigenic variability (eg, Ala'Aldeen & Borriello, (1996) ) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. "Vaccine 14 (1): 49-53).

For meningococcal and gonococcal genes and proteins, a certain amount of sequence data is available (eg. EP-A-0467714 . WO96 / 29412 ), but they are in no way complete. Dempsey et al., (1995) J. Bacteriol. 177: 6390-400, disclosed a physical map for the chromosome of a N. meningitidis strain of serogroup A, and Moxon (1997), Lancet 350: 1240-44, reported that the genome sequences of various pathogens, including N. meningitidis to be determined. The provision of additional sequences could provide a means of identifying secreted or surface-exposed proteins that are the putative targets for the immune system and have no antigenic variability. For example, some of the identified proteins could be components of effective Meningococcus B vaccines, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic Neisseriae, including Neisseria meningitidis or Neisseria gonorrhoeae. Those of N. meningitidis or N. gonorrhoeae specific sequences which are more highly conserved are further preferred sequences.

It is therefore an object of the present invention, DNA sequences of Neisseria encoding proteins which are antigenic or immunogenic activity.

THE INVENTION

The The invention provides proteins comprising the amino acid sequences of N. meningitidis and the amino acid sequences of N. gonorrhoeae disclosed in the examples.

she also provides proteins having sequence homologs (i.e., those having sequence identity) to the amino acid sequences of N. meningitidis disclosed in the examples include and provide protection against disease by Neisseria bacteria. Dependent on from the particular sequence, the degree of homology (sequence identity) is preferred higher than 50% (eg 60%, 70%, 80%, 90%, 95%, 99% or higher). These proteins enclose mutated and allelic variants of the sequences disclosed in the examples. Typically, 50% or higher identity is between two proteins as an indication of functional equivalence considered. identity between proteins is preferred by the search algorithm Homology is determined by Smith-Waterman, as in the MPSRCH program (Oxford Molecular), using a search for affinity gaps the parameters: gap penalty 12, gap extension penalty 1 is used.

The Invention provides about it In addition, proteins comprising fragments of the amino acid sequences of N. meningitidis and the amino acid sequences of N. gonorrhoeae, which are disclosed in the examples. The Fragments should consist of at least n consecutive amino acids include the sequences and n is dependent on the respective Sequence 8 or greater (e.g. B. 10, 12, 14, 16, 18, 20 or greater). The fragments include an epitope from the sequence.

The Proteins of the invention can Naturally by various means (eg recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various ways (eg, native, fusions, etc.). They are preferred prepared in substantially pure or isolated form (i.e. Substantially free of other host cell proteins of N. meningitidis and N. gonorrhoeae).

in the In accordance with another aspect, the invention provides antibodies which to these amino acid sequences tie. these can may be polyclonal or monoclonal and may be in any suitable manner getting produced.

in the In accordance with another aspect, the invention provides nucleic acids which the nucleotide sequences of N. meningitidis and the nucleotide sequences of N. gonorrhoeae and disclosed in the examples become.

In accordance with another aspect, nucleic acids are provided having a sequence identity of greater than 50% (eg, 60%, 70%, 80%, 90%, 95%, 99%, or more) to the sequences given herein on point. Sequence identity is determined as described above.

Nucleic acid, the Also included are fragments of these sequences. These n should be consecutive nucleotides from the sequences of N. meningitidis or the sequences of N. gonorrhoeae, and n is dependent from the respective sequence 12 or larger (eg 20, 25, 30, 35, 40 or larger).

in the In accordance with another aspect, the invention provides nucleic acid which Proteins and protein fragments of the invention encoded.

It it should also be noted that the invention provides nucleic acid, comprises the sequences which are complementary to those described above are (for example for Antisense or probe purposes).

Nucleic acid consistent Of course, with the invention produced in many ways (eg by chemical synthesis, partially or wholly, from genomic or cDNA libraries, from the organism itself, etc.) and can have different forms (eg single-stranded, double-stranded, Vectors, probes, etc.).

In addition, the term "nucleic acid" includes DNA and RNA and also their analogs such as those that modified backbones contain, and also protein nucleic acids (PNA) etc.

in the In accordance with another aspect, the invention provides vectors, the nucleotide sequences of the invention include (e.g., expression vectors), and host cells transformed with such vectors.

in the In accordance with another aspect, the invention provides compositions, the protein, antibodies and / or nucleic acid in accordance with the invention. These compositions can to Example as vaccines or as diagnostic reagents or as immunogenic compositions.

The Invention also provides nucleic acid, Protein or antibody in accordance with the invention for use as a pharmaceutical (e.g. As vaccines) or as diagnostic reagents. She delivers also the use of nucleic acid, Protein or antibody in accordance with the invention for the preparation of (i) a medicament for preventing infection by Neisseria bacteria, (ii) a diagnostic reagent for detection the presence of Neisseria bacteria or of antibodies, which have been formed against Neisseria bacteria, or (iii) Formation of antibodies. The mentioned Neisseria bacteria can be of any kind or belong to any tribe (such as N. gonorrhoeae) but are preferably N. meningitidis, especially serogroup B or serogroup C.

One Method of treating a patient may be administration a therapeutically effective amount of nucleic acid, protein and / or antibody in the Consistent with the invention to the patient.

One Method for producing proteins of the invention is provided which is consistent with the step of cultivating a host cell with the invention under conditions that include expression of the protein.

A Summary of Standard Techniques and Methods Using can be to carry out the invention (eg, the disclosed sequences for vaccination or for diagnostic Purposes) is attached as an annex to the description. In the In summary, examples are given that can be used.

To This general description of the invention is described by the Reference is made to the following examples, given by way of illustration should be better understood.

Methodology - Summary of Standard Procedures and techniques

Generally

This invention provides nucleotide sequences of Neisseria meningitidis menB and amino acid sequences zen, which are coded by it. With these disclosed sequences, assays can be made with nucleic acid probes and expression cassettes and vectors. The expression vectors can be transformed into host cells to produce proteins. The purified or isolated polypeptides (which may also be chemically synthesized) can be used to make antibodies that can be used to detect the human B-protein. Also, the host cells or extracts can be used for biological studies to isolate agonists or antagonists. In addition, one can screen with these sequences to identify open reading frames and to identify amino acid sequences. The proteins may also be used in immunogenic compositions, antigenic compositions and as components of vaccines.

The execution the present invention is, unless indicated otherwise, conventional techniques from molecular biology, microbiology, procedures with recombinant DNA and conventional Use techniques from immunology that contribute to the expertise of each Territories belong. Such techniques are fully explained in the literature, e.g. B. Sambrook, Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.N. Glover, Ed., 1985); Oligonucleotide Synthesis (M.J. Gait Ed., 1984); Nucleic acid Hybridization (B.D. Hames & S. J. Higgins, ed., 1984); Transcription and Translation (B.D. Hames & S.J. Higgins, Eds., 1984); Animal Cell Culture (R.I. Freshney, Ed., 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series Methods in Enzymology (Academic Press Inc.), especially volumes 154 &155; Gene Transfer Vector for Mammalian Cells (J.H. Miller and M.P. Calos, Eds., 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purificafion: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C.C. Blackwell, Eds., 1986).

In This specification uses standard abbreviations for nucleotides and amino acids.

All Publications, Patents and patent applications cited herein are fully incorporated by reference Reference included.

expression systems

The Nucleotide sequences of Neisseria menB can be linked to a number of different Expression systems are expressed; for example, those at Mammalian cells, Plant cells, baculoviruses, bacteria and yeast are used.

i. Mammalian systems

Mammalian expression systems are known in the art. A mammalian promoter is any one DNA sequence capable of binding mammalian RNA polymerase and the downstream directed (3 ') transcription a coding sequence (eg of a structural gene) into mRNA. A promoter becomes a region for initiating transcription, usually proximal to the 5 'end the coding sequence, and a TATA box, usually 25-30 base pairs (bp) upstream the site for initiating transcription is owned. The TATA box is said to control RNA polymerase II, allowing it to target RNA synthesis the correct place begins. A mammalian promoter will also become one upstream contained promoter element usually within 100 to 200 bp upstream the TATA box is located. An upstream promoter element is determined the rate at which transcription is initiated, and may be in any Direction (Sambrook et al., 1989) Expression of Cloned Genes in Mammalian Cells. ' In Molecular Cloning: A Laboratory Manual, 2nd ed.).

Mammalian virus genes are often expressed at high rates and have a wide range of host cells; therefore, sequences encoding mammalian virus genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the major adenovirus late promoter (Ad MLP), and the herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also provide useful promoter sequences. Expression can be either constitutive or regulated (inducible). Depending on the promoter selected, many promoters may be inducible using known substrates, such as the use of the mouse tumor virus (MMTV) promoter with the glucocorticoid-responsive element (GRE), which is induced by glucocorticoid in transformed cells the hormone is induced (see, for example U.S. Patent No. 5,783,681 ).

The Presence of an enhancer element (enhancer) combined with the promoter elements described above, the expression levels usually increase. An enhancer is a regulatory DNA sequence that regulates transcription can stimulate up to 1000 times when using homologs or heterologous promoters, wherein the synthesis of the normal RNA start site starts. Enhancers are also active when they are upstream or downstream downstream the site for initiation of transcription either in normal or in the opposite direction or at a distance of more than 1000 nucleotides from the promoter (Maniatis et al., (1987) Science 30 236: 1237; Alberts et al., (1989) Molecular Biology of the Cell, 2nd ed.). Enhancer elements that come from viruses can be special useful because they are ordinary have another area of hosts. Examples include the early SV40 gene enhancer (Dijkema et al., (1985) EMBO J. 4: 761) and the Enhancers / promoters derived from the long terminal repeat (LTR) of the Rous sarcoma virus (Gorman et al., (1982b) Proc. Natl. Acad. Sci. 79: 6777) and the human cytomegalovirus (Boshart et al., (1985) Cell 41: 521). In addition, some are enhancers regulable and are only in the presence of an inducing agent such as a hormone or metal ion active (Sassone-Corsi and Borelli, (1986) Trends Genet. 2: 215; Maniatis et al., (1987) Science 236: 1237).

One DNA molecule can be in mammalian cells intracellularly are expressed. A promoter sequence can be linked directly to the DNA molecule In this case, the first amino acid will be at the N-terminus of the recombinant Proteins always be a methionine, by the ATG start codon is coded. If desired, can the N-terminus by in vitro incubation with cyanogen bromide from Protein are split off.

alternative can also secretes foreign proteins from the cell into the culture medium be by chimera DNA molecules which encode a fusion protein comprising a leader sequence fragment, which allows the secretion of the foreign protein in mammalian cells. Processing sites between the leader fragment are preferred and the foreign gene, either in vivo or in vitro can be split. The leader sequence fragment usually codes a signal peptide that includes hydrophobic amino acids that regulate secretion control of the protein from the cell. The three-part leader sequence The adenovirus is an example of a leader sequence that causes the secretion of a foreign protein in mammalian cells.

Usually are the sequences for terminating transcription and for polyadenylation, that of mammalian cells be recognized, regulatory regions, the 3'-ward of the translation stop codon and therefore the coding sequence Flank along with the promoter elements. The 3 'terminus of the mature mRNA is characterized by site specific posttranscriptional cleavage and Polyadenylation (Bimstiel et al., (1985) Cell 41: 349; Proudfoot and Whitelaw (1988) "Termination and 3 'end processing of eukaryotic RNA. In Transcription and Splicing (Ed. B.D. Hames and D.M. Glover); Proudfoot, (1989) Trends Biochem. Sci. 14: 105). These sequences control the transcription of an mRNA that enters the Polypeptide which is encoded by the DNA to be translated can. examples for Termination / polyadenylation signals for transcription enclose those derived from SV40 (Sambrook et al., (1989) "Expression of cloned genes in cultured mammalian cells. " Molecular Cloning: A Laboratory Manual).

Usually will the above-described components which contain a promoter Polyadenylation signal and a sequence to terminate transcription enclose, assembled in expression constructions. Enhancers, introns with functional Splice donor and acceptor sites and leader sequences may also be engineered into an expression construct included, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids), the to a stable fate in a host cell such as mammalian cells or bacteria is able. Mammalian replication systems include those derived from animal viruses and the transacting factors need, to replicate. For example, plasmids replicate, containing the replication systems of Papova viruses, such as SV40 (Gluzman, (1981) Cell 23: 175) or the polyoma virus, in the presence of the appropriate viral T-antigen to an extremely high number of Copies. additionally enclose examples for Mammalian replicons those derived from bovine papilloma virus and Epstein-Barr virus descended. additionally For example, the replica may have two replication systems, thereby allowing for example, for expression in mammalian cells and for cloning and amplification in a prokaryotic host organism becomes. examples for such mammalian-bacterial shuttle vectors enclose pMT2 (Kaufman et al., (1989) Mol. Cell. Biol. 9: 946) and pHEBO (Shimizu et al., (1986), Mol. Cell. Biol. 6: 1074).

The transformation method used depends on the host organism that is to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-vermit teltransfection, protoplast fusion, electroporation, inclusion of polynucleotide (s) in liposomes and direct microinjection of the DNA into the nuclei.

Mammalian cell lines, which can be used as host cells for expression are known in the art and include many immortalized cell lines, provided by the American Type Culture Collection (ATCC) be, including, but not limited to this Chinese Hamster Quartz (CHO) cells, HeLa cells, kidney cells of baby hamsters (BHK), monkey kidney cells (COS), carcinoma cells from human liver tissue (eg Hep G2) and a number of others Cell lines.

ii. Expression systems with plant cells

There are many genetic expression systems known in the art with plant cell cultures and whole plants. Exemplary plant cell genetic expression systems include those described in patents, such as US Pat U.S. Patent No. 5,693,506 ; U.S. Patent No. 5,659,122 ; and U.S. Patent No. 5,608,143 , Additional examples of genetic expression in plant cell culture have been described by Zenk, Phytochemistry 30: 3861-3863 (1991). Descriptions of plant protein signal peptides may be made in addition to the references described above in Vaulcombe et al., Mol. Genet. 209: 33-40 (1987); Chandler et al., Plant Molecular Biology 3: 407-418 (1984); Rogers, J. Biol. Chem. 260: 3731-3738 (1985); Rothstein et al., Gene 55: 353-356 (1987); Whittier et al., Nucleic Acids Research 15: 2515-2535 (1987); Wirsel et al., Molecular Microbiology 3: 3-14 (1989); Yu et al., Gene 122: 247-253 (1992). A description of the regulation of plant gene expression by the phytohormone gibberellic acid and secreted enzymes induced by gibberellic acid can be found in RL Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, eds , Pitman Publishing Limited, London, pp. 21-52. References describing other metabolically regulated genes are: Sheen, Plant Cell, 2: 1027, 1038 (1990); Maas et al., EMBO J. 9: 3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84: 1337-1339 (1987).

typically, will be a desired Polynucleotide sequence using art known in the art Techniques inserted into an expression cassette, which genetic Encloses regulatory elements, the for their function has been designed in plants. The expression cassette becomes along with accompanying sequences upstream and downstream the expression cassette used for Expression in a plant host organism are suitable, in a desired Inserted expression vector. The accompanying sequences become plasmidic or of viral origin and have the necessary vector properties, to enable the vectors DNA from an original one Host organism for cloning such as bacteria to the desired to transfer plant host organism. The basic bacterial-herbal vector design becomes prefers a prokaryotic origin of replication with a broad host range, a prokaryotic selectable marker and for the transformations of Agrobacterium, T-DNA sequences for a through Agrobacterium mediated transfer to use on plant chromosomes. Where the heterologous gene is a proof not directly accessible Preferably, the construction also becomes a selectable marker gene have that been used to determine if a plant cell has been transformed is, is suitable. A general overview of suitable markings, for example for Members of the grass family can be found at Wilmink and Dons, 1993, Plant Mol. Biol. Rep. 11 (2): 165-185, find.

sequences the for a permission of the integration of the heterologous sequence into the plant Genome are also recommended. These can be for the homologues Recombination both transposon sequences and the like as well Ti sequences, the insertion of a heterologous expression cassette in randomize a plant genome. suitable Prokaryotic selectable markers enclose resistance across from Antibiotics such as ampicillin or tetracycline. Other DNA sequences, which encode further functions may also be in the vector which is known in the art.

The Nucleic acid molecules of the present Invention can for the Expression of the proteins of interest (of the protein of interest) be included in an expression cassette. Usually only becomes an expression cassette is present, although two or more conceivable are. The recombinant expression cassette is added to the heterologous protein coding sequence is the following Elements include: a promoter region, plant, untranslated 5 'sequences, start codon dependent on of whether the structural gene is already hereby equipped or not, and a sequence to stop transcription and translation. Distinctive places for Restriction enzymes at the 5'- and 3 'ends of the cassette allow easy insertion into an already existing vector.

A heterologous coding sequence can be used for any protein in the Be prepared in accordance with the present invention. The sequence, encoding the protein of interest will encode a signal peptide which the processing and translocation of the protein, if appropriate, and you will usually miss any sequence, the one bond of the desired Protein of the invention to a membrane could result. There in most cases the transcription start region for will be a gene that during The germination phase is expressed and translocated, you can by Use of the signal peptide which causes the translocation also effect the translocation of the protein of interest. To this Way, the protein of interest (the proteins of interest) be translocated from the cells in which it is expressed and can be harvested effectively. Typical secretion of semen occurs via the Aleuron or scutellar epithelial layer into the endosperm of the Seed. Although it is not necessary for the protein to be derived from the Cells in which it is produced, secreted, relieved this, however, the isolation and purification of the recombinant protein.

There the final Expression of the desired Gene product is to take place in a eukaryotic cell is it desirable to determine if any section of the cloned gene sequences contains that of the spliceosome machinery of the host organism are cut out as introns. If so For example, site-directed mutagenesis of the "intron" region can be performed to reduce the loss a section of the genetic message as a false intron code to prevent Reed and Maniatis, Cell 41: 95-105, 1985.

Of the Vector can be achieved by the use of micropipettes for mechanical transmission the recombinant DNA are microinjected directly into plant cells, Crossway, Mol. Gen. Genet, 202: 179-185, 1985. The genetic Material can also be made using polyethylene glycol in the Be transferred to the plant cell, Krens, et al., Nature, 296, 72-74, 1982. Another method for introducing nucleic acid segments is the high-speed ballistic penetration of small particles, wherein the nucleic acid either within the matrix of small beads or particles or on their surface Klein, et al., Nature, 327, 70-73, 1987, and Knudsen and Muller, 1991, Planta, 185: 330-336, teach the particle bombardment of endosperm of barley to transgenic To produce barley. Yet another method of introduction would be fusion protoplasts with other units, either minicells, cells, Lysosomes or other fusible bodies with lipid surface, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

Of the Vector can also be introduced into the plant cells by electroporation (Fromm et al., Proc Natl Acad Sci., USA 82: 5824, 1985). At this Technology become plant protoplasts in the presence of plasmids, which contain the gene construction, subjected to electroporation. High field strength electrical pulses permeabilize biomembranes reversible, what the introduction the plasmids allowed. Plant protoplasts in which one Electroporation performed was, rebuild the cell wall, divide and form one Plant callus.

All Plants from which protoplasts are isolated and cultured can, to produce whole, regenerated plants, can by the present Be transformed invention so that whole plants recovered who transmitted that Gene included. It is known that virtually all plants are cultivated from Cells or tissues can be regenerated, including, but not limited to this all the more important types of sugarcane, sugarbeet, cotton, fruit and other trees, Legumes and Vegetables. Some suitable plants include, for example, species of Genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum and Datura.

Regeneration procedures vary from plant species to plant species, but generally a suspension of transformed protoplasts containing copies of the heterologous gene will generally be prepared. Callus tissue is formed and shoots can be induced from the callus and then rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate like natural embryos to form plants. The culture media will generally contain various amino acids and hormones such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the culture medium, especially in such species as corn and alfalfa. Saplings and roots usually develop at the same time. Effective regeneration will depend on the culture medium, the genotype and the history of the culture. If these three variables ge be controlled, the regeneration is completely reproducible and repeatable.

In Some plant cell culture systems may contain the desired protein excreted in the invention, or alternatively, the protein be extracted from the entire plant. If the desired protein of the invention is secreted into the culture medium, it can be collected become. Alternatively you can the embryos and embryo-free semi-seeds or other plant tissue be mechanically disrupted to secreted protein between Release cells and tissues. The mixture can be suspended in a buffer solution become soluble Recover proteins. It then becomes conventional Method for isolation and purification of protein used to to purify the recombinant protein. The parameters time, temperature, pH, oxygen and volume are adjusted by routine procedures, to optimize expression and recovery of heterologous protein.

iii. Baculovirus systems

The Polynucleotide encoding the protein may also be transformed into a suitable Expression vector of an insect can be inserted and is with the Control elements functionally connected within this vector. For the construction of the vector are used techniques known in the art. General enclose the components of the expression system are usually a transfer vector bacterial plasmid containing both a fragment of the baculovirus genome as well as a common one Restriction site for the Insertion of the heterologous gene or heterologous genes that expresses to be included; a wild-type baculovirus having a sequence similar to that for the baculovirus specific fragment in the transfer vector is homologous (this is allowed the homologous recombination of the heterologous gene into the baculovirus genome in); and suitable insect-derived host cells and culture medium for the Growth.

To the insertion of the DNA sequence encoding the protein into the transfer vector For example, the vector and the wild-type viral genome become an insect-host cell in which the vector and the viral genome are allowed is going to recombine. The packaged recombinant virus is expressed, and recombinant plaques are identified and purified. materials and methods for Baculovirus / insect cell expression systems are commercially available in kit form acquire, inter alia, from Invitrogen, San Diego CA ("MaxBac" kit). These techniques are well known and appreciated by those skilled in the art Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter "Summers and Smith ").

In front the insertion of the DNA sequence encoding the protein into the baculovirus genome are as above described components containing a promoter, a leader sequence (if desired), the coding sequence of interest and a sequence for termination transcription, usually in an intermediate Construction for transmission (Transfer vector) united. This construction can be a single one Gene and functionally linked regulatory elements; several genes, each with its own set of functionally linked regulatory elements; or multiple genes produced by the same set of regulatory elements be regulated. Intermediary constructions for transmission are often in a replicon such as an extrachromosomal element (For example, plasmids), which leads to a stable fate in a Host organism such as a bacterium is able. The replicon will have a replication system that will allow him to for cloning and amplification in a suitable host organism to persist.

Of the most commonly used transfer vector for introducing foreign Gene in AcNPV is currently pAc373. Many other vectors, the professionals are known in the art have also been designed. These enclose for example, pVL985 (which replaces the polyhedrin start codon of ATG ATT changes and which a BamHI cloning site 32 base pairs downstream of Introduces ATT; see. Luckow and Summers, Virology (1989) 17:31).

The Contains plasmid usually also the polyhedrin polyadenylation signal (Miller et al., (1988) Ann. Rev. Microbiol, 42: 177) and a procaryotic gene for ampicillin resistance (amp) and an origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating downstream (5 'to 3') transcription of a coding sequence (e.g., a structural gene) into mRNA , A promoter will have a transcription initiation region, usually proximal to the 5 'end of the coding sequence. This region for initiating transcription usually includes a binding site for RNA polymerase and a site for initiating transcription. A baculovirus transfer vector may also have a second domain, called an enhancer, which, if present, is usually a fragment of the structural gene. Expression can be either regulated or constitutive.

Structural genes that are abundantly transcribed at late times in a viral infection cycle provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedrin protein, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression: in: The Molecular Biology of Baculoviruses (Ed. Walter Doerfler); EPO Publ. No. 127 839 and 155 476 ; and the gene encoding the p10 protein, Vlak et al., (1988) J. Gen. Virol. 69: 765th

DNA, encoding the appropriate signal sequences may be secreted by genes Insect or baculovirus proteins such as the baculovirus polyhedrin gene (Carbonell et al., (1988) Gene 73: 409). Because the signals for post-translational Modifications in mammalian cells (such as signal peptide cleavage, proteolytic cleavage and phosphorylation) of insect cells seem to be recognized and the signals for secretion and accumulation needed at the core apparently, also between the cells of invertebrates and cells of vertebrates are conserved, alternatively also leader sequences used, which are not of insect origin, such as those which are derived from genes, the human alpha (alpha) interferon, Maeda et al., (1985) Nature 315: 592; Human gastrin releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8: 3129; Human IL-2, Smith et al. (1985) Proc. Natl. Acad. Sci. USA, 82: 8404; IL-3 mouse, (Miyajima et al., (1987) Gene 58: 273; and glucocerebrosidase of Humans, Martin et al. (1988) DNA, 7:99, encode secretion to cause insects.

One recombinant polypeptide or polyprotein can be expressed intracellularly or it can, if it expresses with the correct regulatory sequences will be secreted. Good intracellular expression of unfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence, which suitable signals for the initiation of translation included an ATG start signal precede. If desired, can methionine at the N-terminus by in vitro incubation with cyanogen bromide be split off from the mature protein.

alternative can recombinant polyproteins or proteins that are not natural be secreted by the insect cell to be secreted by chimera DNA molecules which encode a fusion protein which is a fragment a leader sequence that involves the secretion of the foreign protein in insects. The fragment of the leader sequence usually encodes Signal peptide, which includes hydrophobic amino acids, the translocation of the protein in the endoplasmic reticulum controls.

To Insertion of the DNA sequence and / or the gene that is the precursor of the Expression product of the protein encoded, an insect-host cell with the heterologous DNA of the transfer vector and the genomic Wild-type baculovirus DNA is cotransformed - usually by co-transfection. The promoter and the sequence for the termination of the transcription of the construction usually becomes one 2-5 kb huge Section of the baculovirus genome. Procedure for the introduction of heterologous DNA to the desired Baculovirus sites are known in the art. (See Summers and Smith, supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3: 2156; and Luckow and Summers (1989)). For example may be insertion into a gene such as the polyhedrin gene double homologous crossover recombination occurs; the insertion can also be in a position for a restriction enzyme which results in the desired baculovirus gene added has been. Miller et al., (1989), Bioessays 4:91. If the DNA sequence cloned in place of the polyhedrin gene in the expression vector will, both from the for Polyhedrin specific 5'- as well as 3 'sequences flanked and becomes downstream of the polyhedrin promoter positioned.

The newly formed baculovirus expression vector is then packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); in this way, the majority of virus produced after co-transfection is still the wild-type virus. Therefore, a method for identifying recombinant viruses is required. An advantage of the expression system is the visual screening that allows to distinguish recombinant viruses. The polyhedrin protein produced by the original virus is produced at very high levels in the nuclei of infected cells at late time points after viral infection. Accumulated polyhedrin protein forms inclusion bodies which additionally contain embedded particles. These inclusion bodies, up to 15 μm in size, are highly refractile, giving them a bright-luster appearance that can easily be detected under the light microscope. Cells infected with recombinant viruses lack such inclusion bodies. To distinguish the recombinant virus from the wild-type virus, the supernatant of the transfection is determined using techniques known to those skilled in the art unicellular layer applied to insect cells to form plaques. These plaques are screened under the light microscope for the presence (indicative of the wild-type virus) or absence (indicative of the recombinant virus) of inclusion bodies. Current Protocols in Microbiology, vol. 2 (Ausubel et al., Eds.), Under 16.8 (Erg.10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for the infection of various insect cells. Recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda and Trichoplusia ni (PCT Publ. WO 89/046699 ; Carbonell et al., (1985) J. Virol. 56: 153; Wright (1986) Nature 321: 718; Smith et al., (1983) Mol. Cell. Biol. 3: 2156; and cf. generally Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25: 225).

cell and cell culture media as well as the direct as well as the fusion expression of heterologous polypeptides in a baculovirus expression system for sale to be acquired; The cell culture technique is one of skill in the art well known. See, for. Summers and Smith, supra.

The modified insect cells can then in a suitable nutrient medium cultured be the stable fate of the plasmid or plasmids, present in the modified insect-host cell, allowed. If the gene for the expression product is under inducible control, the Host cell grown to a high density and expression induced become. Alternatively, if the expression is constitutive, that will Product continuously expressed in the culture medium, and the nutrient medium must be continuously circulated while the product of interest is taken and consumed nutrients added become. The product may be purified by methods such as chromatography, e.g. B. HPLC, affinity chromatography, Ion exchange chromatography, etc., electrophoresis, density gradient centrifugation, solvent extraction or the like. Possibly. In addition, the product can As stated, so to speak, essentially all of Insect-derived proteins, which also secreted into the culture medium be removed or lysed from the insect cells, to provide a product which is at least substantially free residues of the host cell, e.g. Proteins, lipids and polysaccharides, is.

Around To achieve protein expression, recombinant host cells, derived from the transformants, incubated under conditions which allow expression of the sequence encoding the recombinant protein coded. These conditions will depend on the selected host cell vary. The conditions can however, by professionals in the field based on knowledge of the subject area, be easily determined.

iv. Bacterial systems

bacterial Expression techniques are known in the art. A bacterial Promoter is any DNA sequence that is capable of bacterial RNA polymerase too tie and the downstream taking place (3 '-) transcription a coding sequence (eg a structural gene) into mRNA. A promoter will have a region to initiate transcription, usually proximal to the 5 'end the coding sequence is located. The region to initiate transcription surrounds usually a binding site for RNA polymerase and a site for initiating transcription. A bacterial promoter can also be called a second operator domain possessing an adjacent binding site for RNA polymerase, at which RNA synthesis begins, overlaps. The operator allows negatively regulated (inducible) transcription as a gene repressor protein can bind the operator and thus the transcription of a specific Can prevent gene. Constitutive expression may be absent Negative regulatory elements such as the operator occur. additionally can be a positive regulation by a gene activator protein binding sequence can be achieved, which, if present, is usually proximal (5 ') of the binding sequence for RNA polymerase lies. An example for a gene activator protein is the catabolic activator protein (CAP), that helps transcribe the lac operon to Escherichia coli (E. coli) (Raibaud et al., (1984) Annu. Rev. Genet. 18: 173). regulated Expression may therefore be either positive or negative, thereby the transcription amplified either or reduced.

Sequences encoding metabolic metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar degrading enzymes, such as galactose, lactose (lac) (Chang et al., (1977) Nature 198: 1056) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes, such as tryptophan (trp) (Goeddel et al., (1980) Nucl. Acids Res. 8: 4057; Yelverton et al., (1981) Nucl. Acids Res. 9: 731; US Patent No. 4,738,921 ; EPO Publ. No. 036 776 and 121 775 ). The beta-lactamase (bla) promoter system (Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (Ed. I. Gresser)), promoter systems of bacteriophage lambda PL (Shimatake et al. , (1981) Nature 292: 128) and T5 ( U.S. Patent No. 4,689,406 ) also provide useful promoter sequences.

In addition, synthetic promoters that are not found in nature also serve as bacterial promoters. For example, the sequences for activating transcription of the first bacterial or bacteriophage promoter may be joined to the operon sequences of a second bacterial or bacteriophage promoter, thereby producing a synthetic hybrid promoter ( U.S. Patent No. 4,551,433 ). For example, the tac promoter is a hybrid trp-lac promoter which encompasses both the trp promoter and lac operon sequences and which is regulated by the lac repressor (Amann et al., (1983) Gene 25 : 167; de Boer et al., (1983) Proc. Natl. Acad. Sci. 80:21). In addition, a bacterial promoter may include naturally occurring promoters of non-bacterial origin which have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to generate high expression levels for some genes in prokaryotes. The bacteriophage T7 RNA polymerase / promoter system is an example of a coupled promoter system (Studier et al., (1986) J. Mol. Biol. 189: 113; Tabor et al., (1985) Proc. Natl Acad. Sci. 82: 1074). In addition, a hybrid promoter may also comprise a promoter of a bacteriophage and an operator region of E. coli ( EPO Publ. No. 267,851 ).

In addition to a functioning promoter sequence is an effective binding site for a Ribosome also for the expression of foreign genes in prokaryotes of use. In E. coli becomes the binding site for a ribosome called Shine-Dalgarno (SD) sequence and includes a start codon (ATG) and a Sequence with a length from 3-9 Nucleotides that are 3-11 Nucleotides upstream the start codon (Shine et al., (1975) Nature 254: 34). It will believed that the SD sequence involved the binding of mRNA to the ribosome by the mating of bases between the SD sequence and the 3 'end of the 16S rRNA supported by E. coli (Steitz et al., (1979) "Genetic signals and nucleotide sequences in messenger RNA. "In Biological Regulation and Development: Gene Expression (Ed. R. F. Goldberger)). To eukaryotic Genes and prokaryotic genes with a weak binding site for the To express ribosome, it is often necessary to change the distance between to optimize the SD sequence and the ATG of the eukaryotic gene (Sambrook et al., (1989) "Expression of cloned genes in Escherichia coli. "In Molecular Cloning: A Laboratory Manual).

A DNA molecule can be expressed intracellularly. A promoter sequence can be linked directly to the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo or in vitro incubation with a bacterial N-terminal methionine-specific peptidase. EPO Publ. No. 219,237 ).

Fusion proteins provide an alternative to control expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein or other stable protein is fused to the 5 'end of heterologous coding sequences. Upon expression, this construction will provide a fusion of the two amino acid sequences. For example, the cell gene of the lambda bacteriophage can be bound to the 5 'end of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (Factor Xa) to cleave the bacteriophage protein from the foreign gene (Nagai et al. (1984) Nature 309: 810). Fusion proteins may also be linked to lacZ sequences (Jia et al., (1987) Gene 60: 197), trpE- (Allen et al., (1987) J. Biotechnol., 5:93, Makoff et al., (1989). J. Gen. Microbiol. 135: 11) and Chey genes ( EPO Publ. No. 324 647 ) getting produced. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region, which preferably retains a site for a processing enzyme (eg, the ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Natural foreign protein can be isolated by this method (Miller et al., (1989) Bio Technology 7: 698).

Alternatively, foreign proteins may also be secreted by the cell by generating chimeric DNA molecules encoding a fusion protein comprising a signal peptide sequence fragment which promotes secretion of the foreign protein in bacteria ( U.S. Patent No. 4,336,336 ). The signal The sequence fragment usually encodes a signal peptide comprising hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is secreted either into the growth culture medium (Gram-positive bacteria) or into the periplasmic space located between the inner and outer cell membrane (Gram-negative bacteria). Preferably, there are processing sites that can be cleaved either in vivo or in vitro and encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al., (1983) Experimental Manipulation of Gene Expression; Ghrayeb et al., (1984) EMBO J. 3: 2437) and the E. coli alkaline phosphatase signal sequence (PhoA) (Oka et al., (1985) Proc Natl Acad Sci., 82: 7212). As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous B. subtilis proteins (Palva et al., (1982) Proc. Natl. Acad Sci. 5582; EPO Publ. No. 244 042 ).

Usually are the sequences to stop transcription by bacteria be recognized, regulatory regions, the 3'-ward of the translation stop codon and therefore together with the promoter flank the coding sequence. These sequences control transcription an mRNA encoding the polypeptide encoded by the DNA, can be translated. Sequences to terminate transcription enclose often DNA sequences of about 50 nucleotides that are capable of stem-loop structures to form the end of the transcription. Examples enclose Sequences to stop transcription from genes with strong ones Promoters such as the E. coli trp gene as well as other biosynthetic Genes.

Usually will the above-described components which contain a promoter, a Signal sequence (if desired), the coding sequence of interest and the sequence for termination transcription, together in expression constructions united. The expression constructions are often in a replicon such as an extrachromosomal element (e.g., plasmids), which leads to a stable fate in a host organism such as Bacteria are able. The replica becomes a replication system own, which enables him in a prokaryotic host organism is either expressed or to persist for cloning and amplification. In addition, can a replicon is a plasmid of either high or low Be number of copies. A plasmid with a high number of copies Generally, a number of copies will be between about 5 to about 200 and usually about 10 to about 150 have. A host organism that is a plasmid containing a high number of copies is preferred at least 10 and more preferably at least about 20 plasmids. In dependence from the effect of the vector and the foreign protein in the host organism can be a vector with either high or low copy numbers selected become.

Alternatively, the expression constructs can be integrated with an integrating vector into the bacterial genome. Integrating vectors contain at least one sequence that is homologous to the bacterial chromosome that allows integration of the vector. The integrations appear to result from recombinations between the homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome ( EPO Publ. No. 127 328 ). Integrating vectors may also include bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs selectable markers included the selection for bacterial strains that have been transformed are to be allowed. Selectable labels may be present in the bacterial host organism can and can be expressed Include genes, the bacteria are resistant to drugs such as ampicillin, Chloramphenicol, erythromycin, kanamycin (neomycin) and tetracycline (Davies et al., (1978) Annu., Rev. Microbiol., 32: 469). selectable Markers can also include biosynthetic genes, such as those of the Metabolic pathways for the biosynthesis of histidine, tryptophan and leucine.

alternative can some of the components described above in transformation vectors be summarized. Transformation vectors usually include one selectable marker either held in a replicon or developed into an integrating vector as above described.

Expression and transformation vectors, either extrachromosomal replicons or integrate vectors, have been developed for the transformation of many bacteria. For example, expression vectors have been developed for, among others, the following bacteria: Bacillus subtilis (Palva et al., (1982) Proc. Natl. Acad Sci., USA 79: 5582; EPO Publ. No. 036 259 and 063 953 ; PCT Publ. No. WO 84/04541 ), Escherichia coli (Shimatake et al., (1981) Nature 292: 128; Amann et al., (1985) Gene 40: 183; Studier et al., (1986) J. Mol. Biol. 189: 113; EPO Publ. No. 036 776 . 136 829 and 136 907 ), Streptococcus cremoris (Powell et al., (1988) Appl. Environ. Microbiol. 54: 655); Streptococcus lividans (Powell et al., (1988) Appl. Environ. Microbiol. 54: 655), Streptomyces lividans ( U.S. Patent No. 4,745,056 ).

Methods for introducing exogenous DNA into bacterial host cells are well known in the art and usually involve either the transformation of bacteria treated with CaCl ₂ or other agents such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. The transformation methods usually vary with the type of bacteria that is to be transformed. (See, for example, for the use of Bacillus: Masson et al., (1989) FEMS Microbiol., Lett., 60, 273; Palva et al., (1982) Proc. Natl. Acad Sci. 5582; EPO Publ. No. 036 259 and 063 953 ; PCT Publ. No. WO 84/04541 ; for the use of Campylobacter: Miller et al., (1988) Proc. Natl. Acad. Sci. 85: 856; and Wang et al., (1990) J. Bacteriol. 172: 949; for the use of Escherichia coli: Cohen et al., (1973) Proc. Natl. Acad. Sci. 69: 2110; Dower et al., (1988) Nucleic Acids Res. 16: 6127; Kushner (1978) "An improved method for transforming Escherichia coli with ColE1-derived plasmids." In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (HW Boyer and S. Nicosia); Mandel et al., (1970) J. Mol. 53: 159; Taketo (1988) Biochem. Biophys. Acta 949: 318; for the use of Lactobacillus: Chassy et al., (1987) FEMS Microbiol. Lett. 44: 173; for the use of Pseudomonas: Fiedler et al., (1988) Anal. Biochem. 170: 38; for the use of Staphylococcus: Augustin et al., (1990) FEMS Microbiol. Lett. 66: 203; for the use of Streptococcus: Barany et al., (1980) J. Bacteriol. 144: 698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation", in: Streptococcal Genetics (Ed. J. Ferretti and R. Curtiss III); Perry et al., (1981) Infect. Immune. 32: 1295; Powell et al., (1988) Appl. Environ. Microbiol. 54: 655; Somkuti et al., (1987) Proc. 4th Evr. Cong. Biotechnology 1: 412.

v. Expression in yeast

Also Yeast expression systems are known to those skilled in the art. A yeast promoter is any DNA sequence that is capable of yeast RNA polymerase to tie up and down the river taking place (3 ') Transcription of a coding sequence (eg a structural gene) into mRNA. A promoter becomes a region for initiation have transcription which is usually placed proximal to the 5 'end of the coding sequence is. This region for initiating transcription usually includes one Binding site for RNA polymerase (the "TATA box") and a site to initiate transcription. A yeast promoter can also be a second, upstream possessing the active activator sequence (UAS), which, if present, usually distal to the structural gene. The UAS allows regulated (inducible) Expression. Constitutive expression occurs in the absence of a UAS. Regulated expression can be either positive or negative which either enhances or reduces transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore, sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) ( EPO Publ. No. 284 044 ), Enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase and pyruvate kinase (PyK) ( EPO Publ. No. 329 203 ). The yeast PHO5 gene encoding acid phosphatase also provides useful promoter sequences (Myanohara et al., (1983) Proc Natl Acad Sci., USA 80: 1).

In addition, synthetic promoters, which are not found in nature, serve as promoters in yeast. For example, UAS sequences of a yeast promoter can be linked to the region to activate transcription of another yeast promoter, thereby producing a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the region for activating GAP transcription ( U.S. Patent No. 4,876,197 and 4,880,734 ). Other examples of hybrid promoters include promoters consisting of the regulatory sequences of either the ADH2, GAL4, GAL10, or PHO5 gene combined with the region for activating transcription of a gene for a glycolytic enzyme such as GAP or PyK, are composed ( EPO Publ. No. 164 556 ). In addition, a yeast promoter may include naturally occurring promoters of non-yeast origin having the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include (Cohen et al., (1980) Proc. Natl. Acad Sci., USA 77: 1078; Henikoff et al., (1981) Nature 283: 835; Hollenberg et al., (1981). Curr Topics Microbiol Immunol. 96: 119; Hollenberg et al., (1979) "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae," in: Plasmids of Medical, Environmental and Commercial Importance (ed. KN Timmis and A. Puhler); Mercerau-Puigalon et al., (1980) Gene 11: 163; Panthier et al., (1980) Curr. Genet. 2: 109).

One DNA molecule can be intracellular in yeast are expressed. A promoter sequence can directly with the DNA molecule get connected; in this case, the first amino acid will be at the N-terminus always be a methionine produced by the recombinant protein ATG start codon is encoded. If desired, the methionine on the N-terminus cleaved by in vitro incubation with cyanogen bromide become.

Fusion proteins provide an alternative to yeast expression systems, both in mammalian, plant, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein or other stable protein is fused to the 5 'end of heterologous coding sequences. Upon expression, this construction will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene can be joined to the 5 'end of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavage site. See, for. B. EPO Publ. No. 196056 , Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region, which preferably retains a site for a processing enzyme (eg, the ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Thus, native foreign protein can be isolated by this method (e.g. WO88 / 024066 ).

alternative can Foreign proteins also sektretiert from the cell into the culture medium be by chimera DNA molecules which encode a fusion protein which is a fragment a leader sequence that involves the secretion of the foreign protein in yeast ensures. Processing centers are preferred between the leader fragment and the foreign gene, either in can be cleaved in vivo or in vitro. The fragment of the leader sequence usually codes a signal peptide that includes hydrophobic amino acids that regulate secretion control of the protein from the cell.

DNA encoding suitable signal sequences can be obtained from genes for secreted yeast proteins, such as the yeast invertase gene ( EPO Publ. No. 012 873 ; JPO Publ. WO62: 096.086 ) and the A-factor gene ( U.S. Patent No. 4,588,684 ). Alternatively, there are leader sequences of non-yeast origin, such as an interferon leader sequence, which also ensure secretion in yeast ( EPO Publ. No. 060 057 ).

A preferred class of secretory leader sequences are those which utilize a fragment of the alpha-factor gene containing both a "pre" signal sequence and a "pro" region. The types of alpha-factor fragments that can be used include both the full-length pre-pro-alpha factor leader sequence (about 83 amino acid residues) and truncated alpha-factor leader sequences (usually about 25 to about 50 amino acid residues) ( U.S. Patents No. 4,546,083 and 4,870,008 ; EPO Publ. No. 324 274 ). Additional leader sequences utilizing a fragment of the alpha-factor leader sequence which ensures secretion include hybrid alpha-factor leader sequences derived from a pre-sequence of a first yeast but a pro-region of a second yeast alpha factor. (See, for example, PCT Publ. WO 89/02463 .)

Usually are the sequences to terminate the transcription recognized by yeast become regulatory regions that are 3'-wards of the translation stop codon lie together in this way flank the coding sequence with the promoter. These sequences control the transcription of an mRNA encoding the polypeptide, the is encoded by the DNA can be translated. Examples of a sequence to terminate transcription and other yeast-recognized sequences to finish are z. For example, those encoding glycolytic enzymes.

Usually, the components described above, comprising a promoter, a leader sequence (if desired), a coding sequence of interest, and a transcription termination sequence, are grouped into expression constructs. The expression constructs are often maintained in a replicon such as an extrachromosomal element (e.g., plasmids) capable of stably residing in a host organism such as yeast or bacteria. The replicon may have two replication systems, thereby allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host organism for cloning and amplification. Examples of such yeast-bacterial shuttle vectors include YEp24 (Botstein et al., (1979) Gene 8: 17-24), pCI / 1 (Brake et al., (1984) Proc Natl Acad Sci., USA 81: 4642-4646) and YRp17 (Stinchcomb et al., (1982) J. Mol. Biol. 158: 157). In addition, a replicon may be a plasmid with either a high or a low number of copies. A high copy number plasmid will generally have a number of copies ranging from about 5 to about 200, and usually from about 10 to about 150 copies. A host organism containing a high copy number plasmid will preferably have at least about 10 and more preferably at least about 20 copies. Depending on the effect of the vector and foreign protein on the host organism, one may choose between a high or low copy number vector. See Brake et al., Supra.

alternative can the expression constructions with an integrating vector in the genome of the yeast will be integrated. Including integrating vectors usually at least one sequence homologous to a yeast chromosome and which allows the vector to integrate, and preferably two homologous sequences flanking the expression construction. Integrations appear from recombinations between homologous DNA in the vector and chromosome of the yeast (Orr-Weaver et al., (1983) Methods in Enzymol. 101: 228-245). An integrating vector can be directed to a specific site in the yeast by the appropriate homologous sequence for an inclusion in the vector is selected. See Orr-Weaver et al., supra. One or more expression constructs can be integrated that may be affect the levels of recombinant protein produced (Rine et al., (1983) Proc Natl Acad Sci., USA 80: 6750). The chromosomal Sequences that are included in the vector may be either as a single Segment occur in the vector, resulting in the integration of the whole Vector leads, or as two segments homologous to adjacent segments in the chromosome and the expression construction in the vector flank, leading to the stable integration of the expression construct alone to lead can.

Usually, extrachromosomal and integrating expression constructs selectable markers which allow the selection of yeast strains that transform have been. Selectable labels may include biosynthetic genes that in the hips Host cell can be expressed, such as ADE2, HIS4, LEU2, TRP1 and ALG7, and the G418 resistance gene, which the resistance to Tunicamycin or G418 transmits to yeast cells. In addition, a suitable Selectable marker also provide yeast that has the ability has, in the presence of toxic components such as metal too to grow. For example, the presence of CUP1 allows the yeast to to grow in the presence of copper ions (Butt et al., (1987) Microbiol. Rev. 51: 351).

alternative can some of the components described above in transformation vectors be summarized. Transformation vectors usually include one selectable marker either maintained in a replicon or developed into an integrating vector as above described.

Expression and transformation vectors, either as extrachromosomal replicons or as integrating vectors, have been developed for the transformation of numerous yeasts. For example, expression vectors and methods for introducing exogenous DNA into yeast host organisms have been developed, inter alia, for the following yeasts: Candida albicans (Kurtz, et al., (1986) Mol. Cell. Biol. 6: 142); Candida maltosa (Kunze, et al., (1985) J. Basic Microbiol. 25: 141); Hansenula polymorpha (Gleeson, et al., (1986) J. Gen. Microbiol 132: 3459; Roggenkamp et al., (1986) Mol. Gen. Genet. 202: 302); Kluyveromyces fragilis (Das, et al., (1984) J. Bacteriol. 158: 1165); Kluyveromyces lactis (De Louvencourt et al., (1983) J. Bacteriol 154: 737; Van den Berg et al., (1990) Bio / Technology 8: 135); Pichia guillerimondii (Kunze et al., (1985) J. Basic Microbiol. 25: 141); Pichia pastoris (Cregg, et al., (1985) Mol. Cell. Biol. 5: 3376; U.S. Patents No. 4,837,148 and 4,929,555 ); Saccharomyces cerevisiae (Hinnen et al (1978) Proc Natl Acad Sci USA 75: 1929; Ito et al., (1983) J. Bacteriol 153: 163); Schizosaccharomyces pombe (Beach and Nurse, (1981) Nature 300: 706); and Yarrowia lipolytica (Davidow, et al., (1985) Curr Genet 10: 38047, Gaillardin, et al., (1985) Curr Genet. 10:49).

Methods of introducing exogenous DNA into yeast host cells are well known in the art and usually involve either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. The transformation methods usually vary with the yeast species that are to be transformed. See, for. B. [Kurtz et al., (1986) Mol. Cell. Biol. 6: 142; Kunze et al., (1985) J. Basic Microbiol. 25: 141; Candida]; [Gleeson et al., (1986) J. Gen. Microbiol. 132: 3459; Roggenkamp et al., (1986) Mol. Genet. 202: 302; Hansenula]; [Das et al., (1984) J. Bacteriol. 158: 1165; De Louvencourt et al., (1983) J. Bacteriol. 154: 1165; Van den Berg et al., (1990) Bio / Technology 8: 135; Kluyveromyces]; [Cregg et al., (1985) Mol. Cell. Biol. 5: 3376; Kunze et al., (1985) J. Basic Microbiol. 25: 141; U.S. Patents No. 4,837,148 and 4,929,555 ; Pichia]; [Hinnen et al., (1978) Proc. Natl. Acad. Sci. USA 75; 1929; Ito et al., (1983) J. Bacteriol. 153: 163 Saccharomyces); [Beach and Nurse (1981) Nature 300: 706; Schizosaccharomyces]; [Davidow et al., (1985) Curr. Genet. 10:39; Gaillardin et al., (1985) Curr. Genet. 10:49; Yarrowia].

definitions

A Composition containing X, is "essentially free from "Y, if at least 85% by weight of the total X + Y in the composition X represents. Preferably, X comprises at least about 90% by weight of the total X + Y in the composition, more preferably at least about 95% by weight or even 99% by weight.

A "conserved" amino acid fragment or protein from Neisseria is one that is in a particular protein of Neisseria is present in at least x% of Neisseria. The value for x can 50% or more, z. 66%, 75%, 80%, 90%, 95% or even 100% (i.e., the amino acid is found in the protein in question in all Neisseria). Around to determine if an amino acid is "conserved" in a given Neisseria protein, it is necessary to have this amino acid residue in the sequences of the protein in question with a majority of different Neisseria (a reference population). The reference population may include a number of different Neisseria species or can include a single style. The reference population can be a number of different serogroups of a certain kind or a single sero group. A preferred reference population includes the 5 most common Neisseria.

Of the Term "heterologous" refers to two biologicals Ingredients that are not found together in nature. The ingredients can Host cells, genes or regulatory regions such as promoters. Although the heterologous components are not related in nature can be encountered they work together, eg. When a promoter is heterologous to a gene that is functionally linked to the gene. Another An example is when a Neisseria sequence is heterologous to a mouse host cell is.

An "origin of replication" is a polynucleotide sequence, the replication of polynucleotides such as an expression vector initiates and regulates. The origin of replication behaves like an independent one Unit of polynucleotide replication within a cell and is under its own control to replicate in a position. An origin of replication may be needed for a vector so that it replicates in a particular host cell. With certain replication origins may be an expression vector in the presence of the appropriate proteins reproduced within the cell with a high number of copies become. examples for origins of replication they are self-employed replicating sequences that are active in yeast; and the viral T antigen that is effective in COS-7 cells.

A "mutated" sequence is defined as a DNA, RNA or amino acid sequence that is different from, but homologous to, the natural or disclosed sequence. Depending on the particular sequence, the degree of homology (sequence identity) between the natural or disclosed sequence and the mutated sequence is preferably greater than 50% (eg, 60%, 70%, 80%, 90%, 95%). , 99% or greater), which is calculated as described above. As used herein, the term "allelic variant" of a nucleic acid molecule or region for which a nucleic acid sequence is given herein is a nucleic acid molecule or region that occurs substantially at the same site in the genome of another or second isolate and which is genetically modified of natural variation by, for example, mutation or recombination has a similar but not identical nucleic acid sequence. An allelic variant of a coding region typically encodes a protein which has similar activity to that of the protein encoded by the gene to which it is compared. An allelic variant may also involve a change in the 5 'or 3' untranslated regions of the gene, such as in regulatory control regions. (See, for example U.S. Patent No. 5,753,235 ).

antibody

Of the As used herein, "antibody a polypeptide or a group of polypeptides consisting of at least an antibody are constructed combining point. An "antibody combining site" is the three-dimensional one Bonding space with an inner surface shape and charge distribution, the complementary to the properties of an epitope of an antigen are and one Binding of the antibody allow with the antigen. "Antibody" encloses for example antibody of vertebrates, hybrid antibodies, chimeric antibodies, humanized Antibody, changed Antibody, univalent antibodies, Fab proteins and antibodies with a single domain.

Antibodies against the proteins of the invention are for affinity chromatography, an immunoassay and for the discrimination / identification of menB proteins useful by Neisseria. Antibody, which are generated against the proteins of the present invention, bind to antigenic polypeptides or proteins or protein fragments, that are present and specific with menB strains of Neisseria meningitidis are connected. In some cases can these antigens may be associated with specific strains, such as those antigens specific for the menB strains are. The antibodies of the invention immobilized on a matrix and in an immunoassay or in a Column for affinity chromatography used to detect and / or separate polypeptides, Proteins or protein fragments or cells containing such polypeptides, Proteins or protein fragments include. alternative can immobilized such polypeptides, proteins or protein fragments be to antibodies be able to specifically bind to it.

Antibodies against Proteins of the invention, both polyclonal and monoclonal, can by conventional Process are produced. Generally, the protein is first used a suitable animal, preferably a mouse, rat, rabbit or a goat, to immunize. Rabbits and goats are opened Reason for the obtainable volume of serum and the availability of labeled anti-rabbit and anti-goat antibodies for production of polyclonal sera preferred. The immunization becomes common by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant and parenterally injecting the mixture or emulsion (generally subcutaneous or intramuscular) carried out. Typically, a dose of 50-200 μg / injection is sufficient. The Immunization generally becomes 2-6 weeks later preferred with one or more injections of the protein in saline using incomplete Freund's adjuvant refreshed. Alternatively, antibodies can be used in vitro immunization using art known Generate methods for the purposes of this invention are considered equivalent to in vivo immunization becomes. Polyclonal antisera are immunized by blood sampling Add animal to a glass or plastic tube, incubate the blood 25 ° C over a Hour and then Incubation at 4 ° C for 2-18 hours reached. The serum is purified by centrifugation (eg, 1,000 g for 10 minutes). won. About 20-50 ml per decrease can be obtained from rabbits.

monoclonal antibody are prepared using the standard method of Köhler & Milstein (Nature (1975) 256: 495-96) or a modification thereof. Typically will immunize a mouse or rat as described above. Instead of However, the animal will take blood from the animal to take serum Spleen (and optionally several large ones Lymph nodes) and dissociated into individual cells. If desired can the spleen cells are screened (after removal of adherent non specific cells) by placing a cell suspension on a plate or a well coated with the protein antigen is. B cells, the immunoglobulin bound to the membrane, the for the Antigen is specific, express, bind to the plate and become not rinsed with the rest of the suspension. Resulting B-cells or all dissociated spleen cells are then stimulated with myeloma cells to fuse to form hybridomas, and in a selective Culture medium (eg., Hypoxanthine, aminopterin, thymidine culture medium, "HAT") cultivated. The resulting hybridomas are plated by limiting dilution and on the production of antibodies, which specifically bind the immunizing antigen (and which do not bind unrelated antigens). The selected, MAb-secreting hybridomas are then either in vitro (eg. in tissue culture bottles or hollow fiber reactors) or in vivo (such as ascites in mice).

If desired, the antibodies (polyclonal or monoclonal) can be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (especially ³² P and ¹²⁵ I), electron dense reagents, enzymes, and ligands that have specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is commonly detected by its ability to convert 3,3 ', 5,5'-tetramethylbenzidine (TMB) to a blue pigment that is quantifiable by a spectrophotometer. The term "specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, such as in the case of an antigen and a monoclonal antibody specific thereto. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand pairs known in the art. It should be understood that the above description is not intended to assign the various tags to particular classes since the same tag can be used in a number of different ways. For example, ¹²⁵ I may serve as a radioactive label or as an electron-dense reagent. HRP can serve as an enzyme or as an antigen for a mAb. In addition, one can combine different markers for a desired effect. For example, in the practice of this invention, mAK and Avi require din also labels: in this way one could label a mAb with biotin and detect its presence with avidin labeled with ¹²⁵ I or with an anti-biotin mAb labeled with HRP. Other permutations and possibilities will be readily apparent to those skilled in the art, and are considered equivalent in the context of the present invention.

Antigens Immunogens, polypeptides, proteins or protein fragments of the present invention Invention cause the formation of specific binding partner antibodies. These antigens, immunogens, polypeptides, proteins or protein fragments of the present invention include immunogenic compositions of the present invention. Such immunogenic compositions can about that addition adjuvants, carriers or include other compositions which the antigens, polypeptides, proteins or protein fragments of the present invention Support invention or reinforce or stabilize. Such adjuvants and carriers will become those of skill in the art to tap into the field easily.

drug

drug can either polypeptides, antibodies, or nucleic acids of the invention include. The medicines will be a therapeutically effective amount of either polypeptides, antibodies or Polynucleotides of the present invention include.

Of the used herein "therapeutically effective amount " an amount of a therapeutic agent for treatment, relief or prevention of a particular disease or condition Condition or a detectable therapeutic or preventive To show effect. The effect can be for example by chemical Markings or antigen levels are detected. therapeutic Enclose effects also the reduction of physical symptoms such as decreased body temperature after administration to a feverish patient. The exact effective Amount for a patient becomes of the size and the Health status of the patient, the nature and extent of the patient Condition and therapeutic agent or combination of therapeutic Be dependent on the agents selected for administration. It therefore does not make sense to have an accurate effective amount in advance set. The effective amount for a given situation may be however, be determined by routine examinations and is within Discretion of the doctor.

For the purpose The present invention provides an effective dose of about 0.01 mg / kg to 50 mg / kg or from 0.05 mg / kg to about 10 mg / kg of the DNA constructs for the Individual to whom it is administered.

drug may also contain a pharmaceutically acceptable carrier. The term "pharmaceutical compatible Carrier "refers to a carrier for administration of a therapeutic agent such as antibodies or a polypeptide, genes and other therapeutic agents. Of the Expression refers to any pharmaceutical carrier, which does not itself stimulate the production of antibodies to the individual, which receives the composition, Do harm, and without undue toxicity can be administered. Suitable carriers may be large macromolecules, the slow in the body such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Such carriers are professionals on the Area well known.

pharmaceutical compatible Salts can For example, mineral acid salts such as hydrochlorides, Hydrobromides, phosphates, sulfates and the like; and the salts of organic acids, such as acetates, propionates, malonates, benzoates and the like. A Comprehensive discussion of pharmaceutically acceptable excipients is in Remington's Pharmaceutical Sciences (Mack Pub., Co., N.J., 1991).

pharmaceutical compatible excipients in medicines liquids such as water, saline, Glycerin and ethanol included. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like in such carriers available. Typically, the drugs are prepared as injection solutions, either as liquid solutions or suspensions; solid forms intended for solution in or suspension in liquid excipients are suitable before injection can also be prepared become. Liposomes are in the definition of a pharmaceutically acceptable excipient locked in.

delivery methods

As soon as the compositions of the invention have been formulated, they can administered directly to the individual. The individuals to be treated can Be animals; especially human patients can be treated.

direct Administration of the compositions is generally by injection achieved, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or it is administered into the interstitial space of a tissue. The compositions can also be administered in an injury. Other routes of administration enclose oral administration and pulmonary administration, suppositories and transdermal and transcutaneous administrations, needles and gene gunners or hyposprays. The dosage can be over with a treatment plan a single dose or with multiple doses.

vaccines

vaccines in accordance with the invention either prophylactically (i.e., to prevent infection) or therapeutically (i.e., for the treatment of a disease after Infection).

Such Vaccines include immunizing antigen (s) or immunogen (s), immunogenic polypeptide, protein (s) or protein fragments or nucleic acids (e.g. B. ribonucleic acid or deoxyribonucleic acid), usually in association with "pharmaceutical acceptable Carrier ", which is any excipient lock in, which does not itself stimulate the production of antibodies to the individual, which receives the composition, Cause harm. Suitable carriers are typically big Macromolecules that slow in the body such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric Amino acids, Amino acid copolymers, Lipid aggregates (such as oil droplets or Liposomes) and inactive virus particles. Such carriers are available to professionals Well known in the area. In addition, these carriers can act as immunostimulating agents ("adjuvants") For example, the immunogen or antigen may be conjugated to a bacterial toxin such as a toxoid of diphtheria, tetanus, cholera, H. pylori pathogens and the like.

Preferred adjuvants for enhancing the effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc .; (2) Formulations of oil-in-water emulsion (with other specific immunostimulating agents such as muramyl peptides (see below) or components of the bacterial wall or without them) such as, for example, (a) MF59 (PCT Publ. WO 90/14837 containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing but not required various amounts of MTP-PE (see below) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF containing 10% squalane, 0.4% Tween 80, 5% pluronically blocked polymer L121 and thr-MDP (see below), either in U.S. Pat a sub-micron emulsion or vortexed to produce a larger particle emulsion, and (c) Ribi ™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2 squalene, 0.2% Tween 80 and one or more constituents of the bacterial cell wall of the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and the cell wall skeleton (CWS), preferably MPL + CWS (Detox ^™ ); (3) saponin adjuvants such as Stimulon ^™ (Cambridge Bioscience, Worcester, MA) or particles produced therefrom, such as ISCOMs (immunostimulating complexes) may be used; (4) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma Interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc .; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59 are preferred.

As mentioned above Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutam in (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) Etc.

The vaccine compositions containing immunogenic compositions (e.g., which may include the antigen, the pharmaceutically acceptable carrier, and the adjuvant) will typically contain diluents such as water, saline, glycerol, ethanol, etc. In addition, adjuvants such as wetting and emulsifying agents can buffer pH Substances and the like are present in such carriers. Alternatively, vaccine compositions comprising immunogenic compositions may comprise an antigen, polypeptide, protein, protein fragment or nucleic acid in a pharmaceutically acceptable carrier.

More accurate include vaccines comprising immunogenic compositions, both an immunologically effective amount of the immunogenic polypeptides as well as any other of the aforementioned ingredients, if required. "Immunologically effective amount "means that the administration of this amount to an individual, either in a single dose or as part of a series of administrations Treatment or prevention is effective. This amount varies with health and physical Condition of the individual to be treated, the taxonomic group of the individual to be treated (eg non-human primate, Primacy, etc.), the efficiency the immune system of the individual to synthesize antibodies, the Degree of the desired Protection, the formulation of the vaccine, the assessment of the medical situation by the attending physician and others relevant factors. It is expected that the amount in a relative wide range determined by routine examinations can be.

typically, For example, the vaccine compositions or immunogenic compositions are described as injectable substances, either as liquid solutions or suspensions; solid forms intended for solution in or suspension in liquid carriers are suitable before injection can also be prepared become. Drug can also for a reinforced one Adjuvanswirkung be emulsified or enclosed in liposomes, as above for the pharmaceutically acceptable excipients has been discussed.

The immunogenic compositions are usually administered parenterally, z. By injection, either subcutaneously or intramuscularly. additional Formulations for Other modes of administration are suitable, including formulations for oral Administration and pulmonary administration, suppositories and transdermal and transcutaneous applications. The dosage can be over a single dose treatment plan or over a treatment plan with several doses done. The vaccine can be administered in conjunction with other immunoregulatory agents become.

When An alternative to protein based vaccines may be one DNA vaccination performed (eg Robinson & Torres, (1997) Seminar in Immunology 9: 271-283; Donnelly et et. (1997) Annu. Rev. Immunol. 15: 617-648).

Carriers for gene delivery

excipients in gene therapy for delivery of constructions, including a coding sequence of a therapeutic agent of the invention, the to the mammal for expression in the mammal can be delivered be administered either locally or systemically. These constructions can be viral or non-viral vector approaches in in vivo or ex vivo modality use. The expression of such a coding sequence can using endogenous mammalian or heterologous promoters are induced. The expression of the coding Sequence in vivo can be either constitutive or regulated.

The Invention encloses excipients for gene delivery, which are capable of the considered nucleic acid sequences to express. The carrier system for gene delivery is preferably a viral vector and more preferred a retroviral, adenoviral, adeno-associated viral (MV), Herpesvirus or alphavirus vector. The viral vector can also a viral vector derived from astrovirus, coronavirus, orthomyxovirus, Papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus or Togavirus is from. See generally Jolly, (1994) Cancer Gene Therapy 1: 51-64; Kimura, (1994) Human Gene Therapy 5: 845-852; Connelly, (1995) Human Gene Therapy 6: 185-193; and Kaplitt, (1994) Nature Genetics 6: 148-153.

Retroviral Vectors are well known in the art, including the Retroviruses of the type B, C and D, xenotropic retroviruses (for example NZB-X1, NZB-X2 and NZB9-1 (see O'Neill, (1985) J. Virol. 53: 160), polytropic retroviruses, e.g. MCF and MCF-MLV (see Kelly, (1983) J. Virol. 45: 291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985th

sections of Retroviral Gene Therapy Vector can be of various retroviruses come. For example, you can Retrovector LTRs from a murine sarcoma virus, a tRNA binding Place a Rous sarcoma virus, a packaging signal of one murine leukemia virus and an origin for the Synthesis of the second strand derived from a bird leukosis virus.

These recombinant retroviral vectors can be used to generate transduction-competent retroviral vector particles by introducing them into suitable packaging cell lines (cf. U.S. Patent No. 5,591,624 ). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporating a chimeric integrase enzyme into the retroviral particle (cf. WO96 / 37626 ). It is preferred that the recombinant viral vector is a replication-deficient recombinant virus.

Packaging cell lines suitable for use with the retroviral vectors described above are well known in the art and can be readily prepared (see, for example, U.S. Pat. WO95 / 30763 and WO92 / 05266 and can be used to generate producer cell lines (also called vector cell lines or "VCLs") for the production of recombinant vector particles.) Preferably, the packaging cell lines are derived from human parental cells (eg HT1080 cells) or mink-derived Parent cells produced, which precludes inactivation in human serum.

preferred Retroviruses for the construction of retroviral vectors for gene therapy include this Bird Leukemia Virus, Bovine Leukemia Virus, murine leukemia virus, Mink-cell Focus-Inducing Virus, murine sarcoma virus, reticuloendotheliosis virus and Rous sarcoma virus. Particularly preferred murine leukemia viruses enclose 4070A and 1504A (Hartley and Rowe, (1976) J. Virol. 19: 19-25), Abelson (ATCC no. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and the murine Moloney leukemia virus (ATCC No. VR-190). Such retroviruses may be from depositaries or collections such as the American Type Culture Collection ("ATCC") in Rockville, Maryland or from known sources using generally available Techniques are isolated.

Exemplary known gene therapy retroviral vectors that can be used in this invention include those described in the following patent applications: GB2200651 . EP0415731 . EP0345242 . EP0334301 . WO89 / 02468 ; WO89 / 05349 . WO89 / 09271 . WO90 / 02806 . WO90 / 07936 . WO94 / 03622 . WO93 / 25698 . WO93 / 25234 . WO93 / 11230 . WO93 / 10218 . WO91 / 02805 . WO91 / 02825 . WO95 / 07994 . US 5,219,740 . US 4,405,712 . US 4,861,719 . US 4,980,289 . US 4,777,127 . US 5,591,624 , See also Vile, (1993) Cancer Res. 53: 3860-3864; Vile, (1993) Cancer Res. 53: 962-967; Ram, (1993) Cancer Res. 53 (1993) 83-88; Takamiya, (1992) J. Neurosci. Res. 33: 493-503; Baba, (1993) J. Neurosurg. 79: 729-735; Mann, (1983) Cell 33: 153; Cane, (1984) Proc. Natl. Acad. Sci. 81: 6349; and Miller, (1990) Human Gene Therapy 1.

Human adenoviral vectors for gene therapy are also known in the art and can be used in this invention. See, for example, Berkner, (1988) Biotechniques 6: 616 and Rosenfeld, (1991) Science 252: 431, and WO93 / 07283 . WO93 / 06223 and WO93 / 07282 , Exemplary, known adenoviral vectors for gene therapy that may be used in this invention include those described in the above referenced documents and in U.S. Pat WO94 / 12649 . WO93 / 03769 . WO93 / 19191 . WO94 / 28938 . WO95 / 11984 . WO95 / 00655 . WO95 / 27071 ; WO95 / 29993 . WO95 / 34671 . WO96 / 05320 . WO94 / 08026 . WO94 / 11506 . WO93 / 06223 . WO94 / 24299 . WO95 / 14102 . WO95 / 24297 . WO95 / 02697 . WO94 / 28152 . WO94 / 24299 . WO95 / 09241 . WO95 / 25807 . WO95 / 05835 . WO94 / 18922 and WO95 / 09654 have been described. Alternatively, the administration of DNA bound to killed adenovirus as described by Curiel, (1992) Hum. Gene Ther. 3: 147-154. The gene delivery vehicles of the invention also include adenovirus-associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed by Srivastava, WO93 / 09239 , The most preferred AAV vectors include the two inverted AAV terminal repeat sequences in which the natural D sequences have been modified by substitution of nucleotides such that at least 5 natural nucleotides and up to 18 natural nucleotides, preferably at least 10 natural nucleotides and up to 18 natural nucleotides, most preferably 10 natural nucleotides are retained and the remaining nucleotides of the D sequence are deleted or replaced by non-natural nucleotides. The natural D sequences of the inverted AAV terminal repeat sequences are sequences of 20 contiguous nucleotides in each inverted terminal AAV repeat sequence (ie, there is a sequence at each end) that are not involved in HP formation. The non-natural replacement nucleotide can be any nucleotide that does not occur in the natural D sequence in the same position. Other useful exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed by Nahreini, (1993) Gene 124: 257-262. Another example of such an AAV vector is psub201 (see Samulski, (1987) J. Virol. 61: 3096). Another exemplary AAV vector is the double-D ITR vector. The construction of the double-D ITR vector is described in U.S. Patent No. 5,478,745 disclosed. Still other vectors are those made by Carter, U.S. Patent No. 4,797,368 and Muzyczka, U.S. Patent No. 5,139,941 , Chartejee, U.S. Patent 5,474,935 , and Kotin WO94 / 288157 have been disclosed. Yet another example of an AAV vector that can be used in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and controls expression predominantly in the liver. Its structure and construction are disclosed by Su, (1996) Human Gene Therapy 7: 463-470. Additional AAV vectors for gene therapy are described in the patents US 5,354,678 . US 5,173,414 . US 5,139,941 , and US 5,252,479 described.

Gene therapy vectors comprising sequences of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide, such as those described in patents US 5,288,641 and EP0176170 (Roizman). Additional exemplary vectors of the herpes simplex virus include HFEM / ICP6-LacZ, disclosed in U.S. Pat WO95 / 04139 (Wistar Institute), pHSVlac, described by Geller, (1988) Science 241: 1667-1669 and in WO90 / 09441 and WO92 / 07945 , HSV Us3 :: pgC-lacZ, described by Fink, (1992) Human Gene Therapy 3: 11-19, and HSV 7134, 2 RH 105 and GAL4, described in EP0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alphavirus vectors for gene therapy which can be used in this invention. Preferred alphavirus vectors are the Sindbis viruses, Togaviruses, Semliki Forest Virus (ATCC VR-67, ATCC VR-1247), Middleberg Virus (ATCC VR-370), Ross River Virus (ATCC VR). ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923, ATCC VR-1250, ATCC VR-1249, ATCC VR-532), and those described in the U.S. Patent Nos. 5,091,309 . 5,217,879 and WO92 / 10578 to be discribed. In particular, those alphavirus vectors described in US Ser. 08 / 405,627, filed Mar. 15, 1995, WO94 / 21792 . WO92 / 10578 . WO95 / 07994 . US 5,091,309 and US 5,217,879 can be described, usable. Such alpha viruses can be obtained from depositories or collections such as ATCC of Rockville, Maryland, or isolated from known sources using commonly available techniques. Alphavirus vectors with reduced cytotoxicity are preferably used (see USSN 08/679640).

DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. WO95 / 07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors, and most preferably from Sindbis virus vectors.

Other viral vectors suitable for use in the present invention include those derived from the following viruses: poliovirus, for example ATCC VR-58, and those described by Evans, Nature 339 (1989) 385 and Sabin (1973 ) J. Biol. Standardization 1: 115; Rhinovirus, for example ATCC VR-1110 and those described by Arnold, (1990) J. Cell. Biochem. 1401; Poxviruses such as Canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described by Fisher-Hoch, (1989) Proc. Natl. Acad. Sci. 86: 317; Flexner, (1989) Ann. NY Acad. Sci. 569: 86, Flexner, (1990) Vaccine 8:17; in U.S. Patent No. 4,603,112 and U.S. Patent No. 4,769,330 and WO89 / 01973 to be discribed; SV40 virus, for example, ATCC VR-305 and those described by Mulligan, (1979) Nature 277: 108 and Madzak, (1992) J. Gen. Virol. 73: 1533; Influenza virus, for example ATCC VR-797 and recombinant influenza viruses produced by the use of reverse genetic engineering, as in U.S. Patent No. 5,166,057 and by Enami, (1990) Proc. Natl. Acad. Sci. 87: 3802-3805; Enami & Palese (1991) J. Virol. 65: 2711-2713 and Luytjes, (1989) Cell 59: 110 (see also McMichael, (1983) NEJ, Med. 309: 13 and Yap, (1978) Nature 273: 238 and Nature (1979) 277 : 108); Immunodeficiency virus of humans, as in EP-0386882 and von Buchschacher, (1992) J. Virol. 66: 2731; Measles virus, for example ATCC VR-67 and VR-1247 and those in EP-0440219 to be discribed; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern Encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western Encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those derived from Hamre, (1966) Proc. Soc. Exp. Biol. Med. 121: 190.

Delivery of the compositions of this invention into cells is not limited to the aforementioned viral vectors. Other methods and media for delivery can be used, such as nucleic acid expression vectors, polycationic condensed DNA, linked only or unrelated to a killed adenovirus. for example US serial no. 08 / 366,787, filed December 30, 1994, and Curiel (1992) Hum. Gene Ther. 3: 147-154, ligand-linked DNA, cf. for example Wu, (1989) J. Biol. Chem. 264: 16985-16987, eukaryotic carrier cells for delivery, cf. for example US serial no. 08 / 240,030, filed May 9, 1994, and US Ser. 08 / 404,796, deposit of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as in U.S. Patent No. 5,149,655 described, ionizing radiation, as in U.S. Patent No. 5,206,152 and in WO92 / 11033 described, neutralization of nucleic acid loading or fusion with cell membranes. Additional approaches are described by Philip, (1994) Mol. Cell. Biol. 14: 2411-2418 and Woffendin, (1994) Proc. Natl. Acad. Sci. 91: 1581-1585.

By Particle-mediated gene transfer can be used, cf. to the Example US serial no. 60 / 023,867. Recently, the sequence in conventional Vectors, the conventional ones Control sequences for expression in high levels contained, inserted and then incubated with synthetic gene transfer molecules, such as with polymeric DNA-binding cations such as polylysine, protamine and Albumin bound to cell targeting ligands, such as about asialorosomucoid, as described by Wu & Wu, (1987) J. Biol. Chem. 262: 4429-4432, Insulin as described by Hucked, (1990) Biochem. Pharmacol. 40: 253-263, Galactose as described by Plank, (1992) Bioconjugate Chem. 3: 533-539, Lactose or transferrin.

Naked DNA can also be used to transform a host cell. Exemplary naked DNA delivery methods are described in U.S. Pat WO 90/11092 and U.S. Patent No. 5,580,859 described. The efficiency of uptake can be improved by the use of biodegradable latex beads. DNA-coated latex beads are transported into the cells with high efficiency after initiation of endocytosis by the beads. The methods can also be improved by treating the beads to increase hydrophobicity, thereby facilitating the disruption of the endosome and releasing the DNA into the cytoplasm.

Liposomes, which can be used as gene delivery systems, are used in US 5,422,120 . WO95 / 13796 . WO94 / 23697 . WO91 / 14445 and EP-524,968 described. As in USSN. 60 / 023,867, in non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors containing conventional control sequences for expression at high levels, and then incubated with synthetic molecules for gene transfer, such as polymeric DNA. binding cations such as polylysine, protamine and albumin bound to cell-targeting ligands such as asialorosomucoid, insulin, galactose, lactose or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously active promoters. Other non-viral delivery suitable for use includes mechanical delivery systems such as the approach described by Woffendin et al. (1994) Proc. Natl. Acad. Sci. USA 91 (24): 11581-11585. In addition, the coding sequence and the product of its expression can be delivered by the deposition of photopolymerized hydrogel materials. Other conventional methods of gene delivery that can be used to deliver the coding sequence include, for example, the use of hand-held gene transfer particle guns, as in US Pat U.S. Patent No. 5,149,655 described; the use of ionizing radiation to activate the transferred gene as in the patents US 5,206,152 and WO92 / 11033 described.

Exemplary liposome and polycationic gene delivery systems are those described in the U.S. Patents US 5,422,120 and 4,762,915 ; in WO95 / 13796 ; WO94 / 23697 ; and WO91 / 14445 ; in EP-0524968 ; and by Stryer, Biochemistry, pp. 236-240 (1975) WH Freeman, San Francisco; Szoka, (1980) Biochem. Biophys. Acta 600: 1; Bayer, (1979) Biochem. Biophys. Acta 550: 464; Rivnay, (1987) Meth. Enzymol. 149: 119; Wang, (1987) Proc. Natl. Acad. Sci. 84: 7851; Plant, (1989) Anal. Biochem. 176: 420.

A Polynucleotide composition may be the therapeutically effective amount a carrier for gene therapy within the above definition. For the purpose The present invention provides an effective dose between about 0.01 mg / kg to 50 mg / kg or 0.05 mg / kg to about 10 mg / kg of the DNA constructs in the individual to whom it is administered.

delivery methods

As soon as they can be formulated the polynucleotide compositions of the invention are administered: (1) the individual directly; (2) as ex vivo delivery to cells that come from the individual; or (3) in vitro for the expression of recombinant proteins. The individuals to be treated may be mammals or birds. Even humans can be treated.

direct Delivery of the compositions will generally be by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or it is released into the interstitial space of a tissue. The Compositions can also be administered in a tumor or an injury. Other Enclose routes of administration oral administration and pulmonary administration, suppositories and transdermal applications, needles and gene guns or hyposprays. The dosage can after a treatment plan with a single dose or a Treatment plan with multiple doses done.

Methods for ex vivo delivery and reimplantation of transformed cells into an individual are known in the art and are described e.g. In WO93 / 14778 described. Examples of cells useful for ex vivo applications include, for example, stem cells, particularly hematopoietic, lymphoid cells, macrophages, dendritic cells or tumor cells.

Generally can be the delivery of nucleic acids as well as ex vivo- as well as for in vitro applications are carried out by the following methods, for example, dextran-mediated transfection, calcium phosphate precipitation Polybren mediated transfection, protoplast fusion, electroporation, Encapsulation of the polynucleotide / polynucleotides in liposomes and direct microinjection of the DNA into the nuclei; all procedures are well known in the art.

Polynucleotide and polypeptide drugs

In addition to the pharmaceutically acceptable, carriers described above and salts can the following additional Agents with polynucleotide and / or polypeptide compositions be used.

A. Polypeptides

One Examples are polypeptides that include, without limitation, asiolorosomucoid (ASOR); Transferrin; Asialglycoproteine; Antibody; Antibody fragments; ferritin; interleukins; interferons, Granulocyte-macrophage colony stimulating factor (GM-CSF), Granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating Factor (M-CSF), stem cell factor and erythropoietin. Viral antigens like envelope proteins can also be used. Also proteins from other invasive Organisms such as the 17 amino acid peptide of Circumsporozoite protein from Plasmodium falciparum, known as RII, can be used.

B. Hormones, Vitamins etc.

Other Groups that can be included are, for example: hormones, Steroids, androgens, estrogens, Thyroid hormone or vitamins, folic acid.

C. Polyalkylenes, polysaccharides, etc.

Also Polyalkylene glycol may be combined with the desired polynucleotides or Polypeptides are included. In a preferred embodiment is polyalkylene glycol is a Polyethlylenglycol. In addition, mono, Di- or polysaccharides are included. In a preferred embodiment this aspect is the polysaccharide dextran or DEAE-dextran. Also chitosan and poly (lactide-co-glycolide).

D. lipids and liposomes

The desired Polynucleotide or polypeptide may also be included in lipids or before delivery to the individual or to cells that derived from this, are packaged in liposomes.

Inclusion in a lipid is generally accomplished using liposomes that are capable of stably binding or entrapping and retaining nucleic acid. The ratio of condenses The polynucleotide or polypeptide to the lipid preparation may vary, but will generally be about 1: 1 (mg DNA: micromoles of lipid) or have a higher lipid content. For an overview of the use of liposomes as carriers for the delivery of nucleic acids cf. Hug and Sleight, (1991) Biochim. Biophys. Acta. 1097: 1-17; Straubinger (1983) Meth. Enzymol. 101: 512-527.

Liposome preparations for Use in the present invention include cationic ones (positively charged), anionic (negatively charged) and neutral Preparations. It has been shown that cationic liposomes reduce intracellular delivery of plasmid DNA (Felgner, (1987) Proc Natl Acad Sci., USA 84: 7413-7416); mRNA (Melone, (1989) Proc Natl Acad Sci., USA 86: 6077-6081); and purified transcription factors (Debs, (1990) J. Biol. Chem. 265: 10189 to 10192) in a functional form.

Cationic liposomes are available directly. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin from GIBCO BRL, Grand Island, NY. (See also Felgner, supra). Other commercially available liposomes include Transfectace (DDAB / DOPE) and DOTAP / DOPE (Boehringer). Other cationic liposomes can be prepared from directly available materials using techniques well known in the art. See, for. Szoka, (1978) Proc. Natl. Acad. Sci. USA 75: 4194-4198; WO90 / 11092 for a description of the synthesis of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane) liposomes.

In similar Way, anionic and neutral liposomes are directly available, such as from Avanti Polar Lipids (Birmingham, AL), or can be lightweight using directly available Materials are produced. Such materials include other phosphatidylcholine, cholesterol, phosphatidylethanolamine, Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), Dioleoylphoshatidylethanolamine (DOPE). These materials can also with the DOTMA and DOTAP starting materials in suitable proportions be mixed. Process for the preparation of liposomes under Use of these materials are well known in the art.

The Liposomes can multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) or big unilamellar ones Vesicles (LUVs) include. The different liposome-nucleic acid complexes are made using methods known in the art are manufactured. See, for. B. Straubinger, (1983) Meth. Immunol. 101: 512-527; Szoka, (1978) Proc. Natl. Acad. Sci. USA 75: 4194-4198; Papahadjopoulos, (1975) Biochim. Biophys. Acta 394: 483; Wilson, (1979) Cell 17:77); Deamer & Bangham, (1976) Biochim. Biophys. Acta 443: 629; Ostro, (1977) Biochem. Biophys. Res. Commun. 76: 836; Fraley, (1979) Proc. Natl. Acad. Sci. USA 76: 3348); Enoch & Strittmatter, (1979) Proc. Natl. Acad. Sci. USA 76: 145; Fraley, (1980) J. Biol. Chem. (1980) 255: 10431; Szoka & Papahadjopoulos, (1978) Proc. Natl. Acad. Sci. USA 75: 145; and Schaefer-Ridder, (1982) Science 215: 166.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide or polypeptide to be delivered. examples for Lipoproteins that can be used include: chylomicrons, HDL, IDL, LDL and VLDL. Mutants, fragments or fusions of these Proteins can also be used. Also modifications of naturally occurring Lipoproteins can used, such as acetylated LDL. These lipoproteins can direct the delivery of polynucleotides to cells, the lipoprotein receptors expimieren. When lipoproteins include the polynucleotide to be delivered no other targeted ligand prefers the composition locked in.

Naturally occurring Lipoproteins include a lipid and a protein portion. The Protein sections are known as apoproteins. At the moment they are Apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins that interact with the Roman Numbers AI, AII, AIV; CI, CII, CIII.

One Lipoprotein may comprise more than one apoprotein. For example, includes that, of course occurring chylomicron A, B, C and E, with time lose this Lipoproteins A and win the apoproteins C and E. VLDL includes the apoproteins A, B, C, and E, LDL comprise the apoprotein B; and HDL includes the apoproteins A, C and E.

The amino acids of these apoproteins are known and are for example by Breslow, (1985) Annu. Rev. Biochem. 54: 699; Law, (1986) Adv. Exp. Med. Biol. 151: 162; Chen, (1986) J. Biol. Chem. 261: 12918; Kane, (1980) Proc. Natl. Acad. Sci. USA 77: 2465; and Utermann, (1984) Hum. Genet. 65: 232 described.

lipoproteins contain a variety of lipids, including triglycerides, cholesterol (free and esters) and phospholipids. The composition of the lipids varies in course occurring lipoproteins. For example, chylomicrons include predominantly Triglycerides. A more detailed description of the lipid content of naturally occurring Lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986) become. The composition of the lipids is selected so that they participate in the conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen in the manner that hydrophobic interaction and association with the polynucleotide binding molecule is relieved.

Naturally occurring Lipoproteins can For example, be isolated by ultracentrifugation from the serum. Such methods are described in Meth. Enzymol. (Supra); Pitas, (1980) J. Biochem. 255: 5454 to 5460 and Mahey, (1979) J. Clin. Invest. 64: 743-750.

lipoproteins can also in vitro or by recombinant methods by expression the apoprotein genes in a desired Host cell are produced. See, for example, Atkinson, (1986) Annu. Rev. Biophys. Chem. 15: 403 and Radding, (1958) Biochim. Biophys. Acts 30: 443.

lipoproteins can also for sale acquired from, for example, Biomedical Techniologies, Inc., Stoughton, Massachusetts, USA.

A further Description of lipoproteins can be found in Zuckermann et al., PCT application No. US97 / 14465 can be found.

F. Polycationic agents

polycationic Agents can, with or without lipoprotein, in a composition with the desired Polynucleotide or polypeptide may be included for delivery.

polycationic Agents typically have a net positive charge at physiological relevant pH and are able to charge the electrical charge of nucleic acids to neutralize the delivery to a desired location. For this Agents exist in both in vitro, ex vivo and in vivo applications. polycationic Agents can used to nucleic acids intramuscular, subcutaneous etc. to a living individual.

The The following compounds are examples of useful polypeptides as polycationic Agents: polylysine, polyarginine, polyornithine and protamine. Other Enclose examples Histones, protamines, human serum albumin, DNA binding proteins, chromosomal non-histone proteins, coat proteins of DNA viruses such as (X174, Transcription factors also contain domains that bind DNA, and therefore can as nucleic acid condensing agents useful be. Shortly, Transcription factors such as C / CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP and TFIID basic domains, bind the DNA sequences.

organic polycationic agents include spermine, spermidine and Purtrescin.

The Dimensions and physical properties of a polycationic Agent can from the above list are extrapolated to other polycationic To construct polypeptide agents or synthetic polycationic Produce agents.

Synthetic polycationic agents

synthetic polycationic agents that are useful are, enclose for example DEAE-dextran, polybrene. Lipofectin and Lipofectamine are monomers that form polycationic complexes when combined with Polynucleotides or polypeptides are combined.

Immunodiagnostic examinations

Neisseria antigens of the invention can be used in immunoassays to detect antibody levels (or vice versa, anti-Neisseria antibodies can be used to detect antigen levels). Immunoassays based on well-defined recombinant antigens can be developed to replace invasive diagnostic procedures. Antibodies to Neisseria proteins in biologi samples, including, for example, blood or serum samples, can be detected. The construction of such immunoassays is subject to great variations and a variety of them are known in the art. For example, protocols for the immunoassay may be based on competition or direct response or sandwich type tests. Protocols may also use, for example, solid supports or may be an immunoprecipitation. Most studies include the use of a labeled antibody or polypeptide; the labels can be, for example, fluorescent, chemiluminescent, radioactive or dye molecules. Studies that amplify the signals of a probe are also known; Examples include studies using biotin and avidin and enzyme-labeled and enzyme-mediated immunoassays, such as ELISA assays.

Kits, the for immunodiagnostic purposes are suitable and the required marked Containing reagents are made by adding the required materials, including the compositions of the invention, in suitable containers along with the rest Reagents and materials (for example, suitable buffer solutions, salt solutions etc.), which for the implementation of the test, as well as a suitable user manual to carry out of the test.

Nucleic acid hybridization

"Hybridization" refers to the attachment of two nucleic acid sequences to each other by hydrogen bonding. Typically, a sequence is fixed on a solid support and the other one will be free in solution. Then be the two sequences under conditions that hydrogen bond favor, be brought into contact with each other. Factors affecting this bond affect, enclose: Type and volume of the solvent; Reaction temperature; time for the hybridization; Move; Agents for blocking the not specific attachment of the sequence from the liquid phase to the solid support (Denhardt's reagent or BLOTTO); Concentration of the sequences; use of Compounds for increasing the rate of assembly of the sequences (Dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al., [Supra] Volume 2, Chapter 9, pages 9.47 to 9.57.

"Stringency" concerns the conditions in a hybridization reaction, the co-storage of very similar Opposite sequences Sequences that differ, prefer. For example, the one should Combination of temperature and salt concentration are chosen which is about 120 to 200 ° C below the calculated Tm of the hybrid to be examined. The Conditions for Temperature and salinity can often determined empirically in previous experiments, in samples of genomic DNA immobilized on filters, be hybridized to the sequence of interest and then under Conditions are washed with different stringencies. See. Sambrook et al., Page 9.50.

Variables that must be considered when, for example, a Southern blot is to be performed are (1) the complexity of the DNA used in the blot and (2) the homology between the probe and the sequences to be detected. The total amount of fragment (s) to be tested may be an order of 10, from 0.1 to 1 μg for plasmid or phage digestion to 10 ^-9 to 10 ^-8 g for a single copy gene in a highly complex eukaryotic Genome vary. For lower complexity polynucleotides, significantly shorter blotting, hybridization and exposure times, less starting polynucleotides, and lower specific activity of the probes can be used. For example, a yeast gene can be detected in single copy with an exposure time of only 1 hour, with 1 μg of yeast DNA starting material, blotting for 2 hours and hybridization for 4-8 hours with a probe at 108 ZpM / μg. For a single-copy mammalian gene, a conservative approach would start with 10 μg of DNA, blotting overnight and hybridizing overnight in the presence of 10% dextran sulfate using a probe of greater than 10 ⁸ zpm / μg, resulting in an exposure time of ~ 24 hours leads.

Numerous factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and thus the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous with the fragment. Other commonly considered variables include the length and total content of G + C of the hybridizing sequences and the ionic strength and formamide content in the hybridization buffer solution. The effects of all these factors can be estimated using a single equation: Tm = 81 + 16.6 (log 10 Ci) + 0.4 [% (G + C)] - 0.6 (% formamide) - 600 / n - 1.5 (% mismatch), where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified according to Meinkoth & Wahl, (1984) Anal Biochem 138: 267-284).

at In the construction of a hybridization experiment, several factors can be identified Hybridization of nucleic acids influence, suitably changed become. The temperature of the hybridization and the washings and the salt concentration during the washings are the easiest to adjust. With increasing temperature of hybridization (i.e., stringency), hybridization becomes less likely between strands occurs that are not homologous, and as a result, the Background. If the radiolabeled probe is not completely homologous to the immobilized fragment (which in the case of the gene family and interspecies hybridization experiments is common), the hybridization temperature must be be lowered, and the background is increasing. The temperature of the washes affects the intensity the hybridizing band and the degree of the background in similar Wise. The stringency of the washes is also increased by decreasing salt concentrations.

Generally be ordinary Hybridization temperatures in the presence of 50% formamide 42 ° C for a probe, which is 95% to 100% homologous to the target fragment, 37 ° C for 90% to 95% homology and 32 ° C for 85% to 90% homology. For lower homologies, the content of formamide should be reduced and the Temperature accordingly, using the above equation, be adjusted. When the degree of homology between the probe and the target fragment is unknown, is the simplest approach, both with hybridization as also to start with washing conditions that are not stringent. If not specific gangs or strong background after the Autoradiography can be observed, the filter can be under high Stringency washed and exposed again. If that for the exposure time required makes this approach impractical, should several Stringencies for the hybridization and / or washing processes are tested in parallel.

Studies with nucleic acid probes

With Methods such as PCR, studies with branched DNA probes or blot techniques in which nucleic acid probes are consistent with can be used in the invention, the presence of cDNA or mRNA can be determined. It is true that a probe with a sequence of the invention "hybridizes" when a Duplex or double-stranded Complex that is stable enough to be detected can be.

The Nucleic acid probes will hybridize to the nucleotide sequences of Neisseria of the invention (including both the sense and antisense strands). Although many different nucleotide sequences are the amino acid sequence will code, that will of course occurring sequence of Neisseria preferred because they are the current in the cells present sequence. The mRNA represents a coding sequence, and therefore a probe should be complementary to the be coding sequence; stranded cDNA is complementary to the mRNA, and therefore a cDNA probe should be complementary to the non-coding sequence.

The Probe sequence need not interfere with the sequence of Neisseria (or its Complement) - some Variations in sequence and length can too high Test sensitivity to lead, when the nucleic acid probe a duplex complex can form with target nucleotides that can be detected. The nucleic acid probe can also be extra Connect nucleotides to stabilize the duplex complex formed. A additional Sequence of Neisseria can also be helpful as a marker to detect the formed duplex complex. For example, can a non-complementary one Nucleotide sequence to the 5 'end attached to the probe with the remainder of the probe sequence being complementary to a Neisseria sequence is. Alternatively you can not complementary Bases or longer Sequences can be interspersed in the probe, provided that the Probe sequence a sufficient complementarity to the Neisseria sequence to hybridize with it, thereby forming a duplex complex, which can be detected.

The exact length and sequence of the probe is determined by the hybridization conditions depending on temperature, salt conditions and the like. To the Example includes the nucleic acid probe for diagnostic Dependent applications from the complexity the sequence to be analyzed is typically at least 10-20 nucleotides, preferably 15-25 and more preferably at least 30 nucleotides, although shorter can. Short primers generally require cooler temperatures to suffice to form stable hybrid complexes with the template.

The Probes can by synthetic methods such as the triester method of Matteucci et al., [J. At the. Chem. Soc. (1981) 103: 3185] or after Urdea et al., [Proc. Natl. Acad. Sci. USA (1983) 80: 7461] or under Use of commercially available to be acquired automated apparatus for oligonucleotide synthesis become.

The chemical nature of the probe can be selected according to preference. For certain Applications is DNA or RNA suitable. For other applications may be modifications be included, for. B. can Modifications to the backbone such as phosphorothioates or methylphosphonates, to increase the in vivo half-life, change RNA affinity, resistance across from Increase nuclease etc. [cf. z. B. Agrawal & Iyer (1995) Curr. Opin. Biotechnol. 6: 12-19; Agrawal (1996) TIBTECH 14: 376-387]; Analogs such as peptide nucleic acids can also be used [see. z. Corey (1997) TIBTECH 15: 224-229; Buchardt et al., (1993) TIBTECH 11: 384-386].

An example of a nucleotide hybridization assay is described by Urdea et al. in the international patent application WO92 / 02526 described [cf. also US Patent246).

Alternatively, the polymerase chain reaction (PCR) is another well known means of detecting small amounts of target nucleic acids. The study is described in: Mullis et al., [Meth. Enzymol. (1987) 155: 335-350]; U.S. Patent No. 4,683,195 ; and U.S. Patent No. 4,683,202 , Two "primer" nucleotides hybridize to the target nucleic acid and are used to initiate the reaction. The primers may comprise a sequence that does not hybridize to the target sequence of the amplification (or its complement) to aid in the stability of the duplex complex or, for example, to include a common restriction site. Typically, such a sequence will flank the desired sequence of Neisseria.

A thermostable polymerase produced using the original Target nucleic acids as a template from the primers copies the target nucleic acids. After this generates a threshold amount of target nucleic acids from the polymerase has been able to these have been detected by more traditional methods such as Southern blots become. If the Southern Blot method is used, the labeled probe with the sequence of Neisseria (or its complement) hybridize.

Also mRNA or cDNA can be detected by traditional blotting techniques by Sambrook et al., [supra]. mRNA or cDNA under Use of a polymerase enzyme has been generated from mRNA, can be purified and separated using gel electrophoresis become. The nucleic acids on the gel are then transferred to a solid support such as nitrocellulose. The solid pad is exposed to a labeled probe and then washed to remove unhybridized probe material. Next the duplex complexes containing the labeled probe are detected. Typically, the probe is labeled with a radioactive moiety.

EXAMPLES

• • eine Nucleotidsequenz, die in N. meningitidis identifiziert worden ist
• • das mutmaßliche Translationsprodukt der genannten Sequenz von N. meningitidis
• • eine zugehörige Nucleotidsequenz, die in N. gonorrhoeae identifiziert worden ist
• • das mutmaßliche Translationprodukt der genannten Sequenz von N. gonorrhoeae
• • einen Vergleich des Prozentsatzes an Identität zwischen dem Translationsprodukt der Sequenz von N. meningitidis und der Sequenz von N. gonorrhoeae

The examples describe nucleic acid sequences identified in N. meningitidis and N. gonorrhoeae, together with their corresponding and putative translation products. The examples include:

• A nucleotide sequence that has been identified in N. meningitidis
• The putative translation product of said N.meningitidis sequence
• An associated nucleotide sequence identified in N. gonorrhoeae
• The putative translation product of said sequence of N. gonorrhoeae
• A comparison of the percentage of identity between the translation product of the sequence of N. meningitidis and the sequence of N. gonorrhoeae

The Sequence comparisons were made at NCBI (http://www.ncbi.nlm.nih.gov) Using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx & tBLASTx carried out [see. also Altschul et al., (1937) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 2289-3402]. Screenings were compared the following databases: non-redundant sequences from GenBank + EMBL + DDBJ + PDB and non-redundant sequences GenBank CDS translations + PDB + SwissProt + SPupdate + PIR.

Lower case letters represent ambiguities that occurred during the alignment of independent sequencing reactions (some of the nucleotide sequences in the examples come from the combination of results from two or more experiments).

The Nucleotide sequences were scanned in all six reading frames the presence of hydrophobic domains using a To predict algorithm based on the statistical investigations by Esposti et al., [Critical Evaluation of the hydropathy of membrane Proteins (1990), Eur. J. Biochem. 190: 207-219). These domains represent potential transmembrane regions or hydrophobic leader sequences.

Open Reading frames were generated using the program ORFFINDER (NCBI) from fragmented nucleotide sequences.

Underlined amino acid sequences show possible Transmembrane domains or leader sequences in the ORFs, as by the PSORT algorithm (http://www.psort.nibb.ac.jp) is predicted. Functional domains were using the MOTIFS program (GCG Wisconsin & PROSITE) as well predicted.

based on the presence of a suspected Leader sequence and / or multiple putative transmembrane domains (single underlined) in the gonococcal protein, it is predicted that the proteins of N. meningitidis and N. gonorrhoeae and their corresponding Epitopes useful Antigens or immunogenic compounds for vaccines or diagnostic Could be means.

The Standard techniques and methods that can be used to to carry out the invention (eg, the use of the disclosed sequences in vaccines or for diagnostic purposes) have been summarized above. These Summary is not a limitation of the invention but is instead Examples that can be used but not required are.

in the Specifically, the following procedures were used to identify the proteins of the invention to express, purify and biochemically characterize.

Preparation of chromosomal DNA

N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC culture medium, harvested by centrifugation and adjusted to pH 8.0 in 5 ml of buffer solution (20% sucrose, 50 mM Tris-HCl, 50 mM EDTA ) resuspended. After incubation on ice for 10 minutes, the bacteria were lysed by adding 10 ml of lysing solution (50 mM NaCl, 1% Na-sarcosyl, 50 μg / ml proteinase K) and the suspension was incubated at 37 ° C for 2 hours. Two phenol extractions (equilibrated to pH 8) and a ChCl ₃ / isoamyl alcohol (24: 1) extraction were performed. DNA was precipitated by addition of 0.3 M sodium acetate and 2 volumes of ethanol and collected by centrifugation. The precipitate was washed once with 70% ethanol and redissolved in 4 ml buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 8). The DNA concentration was measured by reading the CD at 260 nm.

Oligonucleotide design

synthetic Oligonucleotide primers were based on the coding sequence Each ORF is designed by (a) the Meningococcus B sequence, if available, or (b) the Gonococcus / Meningococcus A sequence, adapted to the Meningococcus preferred codon usage were used. All expected signal peptides were removed by the primer sequence for amplification of the 5'-end immediately downstream derived from the expected leader sequence.

For most ORFs, the 5 'primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-NheI or EcoRI-NheI, depending on the restriction pattern of the gene); the 3 'primers included an XhoI restriction site. This method was used to direct the cloning of each individual amplification product (relative to each ORF) in two different expression systems: pGEX-KG (using either BamHI-XhoI or EcoRI-XhoI) and pET21b + (using either NdeI -XhoI or NheI-XhoI).

For some ORFs, two different amplifications were performed to clone each ORF in the two expression systems. Two different 5 'primers were used for each ORF; the same 3'-XhoI primer was used as before:

Other ORFs can be cloned into the expression vector pTRC and expressed as a His-tagged fusion product at the amino terminus. The expected signal peptide may be included in the final product. The NheI-BamHI restriction sites were included using the following primers:

In addition to the restriction enzyme recognition sequences, the primers included nucleotides that hybridized to the sequence to be amplified. The number of hybridizing nucleotides was dependent on the melting temperature of the entire primer and was determined for each primer using the following formula: T m = 4 (G + C) + 2 (A + T) (tail excluded) T m = 64.9 + 0.41 (% GC) - 600 / N (complete primer)

The average melting temperature of the selected oligos was 65-70 ° C for the complete oligo and 50-55 ° C for the hybridizing Region alone.

The oligos were synthesized by a Perkin-Elmer 394 DNA / RNA Synthesizer, eluted from the columns in 2 ml of NH ₄ -OH, and the protecting groups were removed by incubation at 56 ° C for 5 hours. The oligos were precipitated by adding 0.3 M Na-acetate and 2 volumes of ethanol. The samples were then centrifuged and the precipitates resuspended in either 100 μl or 1 ml of water. The OD ₂₆₀ was determined using a Perkin Elmer Lambda Bio-Spectrophotometer and the concentration was determined and adjusted to 2-10 pmol / μl.

If the first amplifications are performed, it may be that the complete 5 'and / or 3' sequence for some ORFs of meningococci is not known, although it may be that identifies the corresponding sequences for gonococcus have been. For the amplification could hence the sequences of gonococcus as the basis for the design from primers in changed Shape to the codon preference Be taken into account. In detail, the following codons are exchanged: ATA → ATT; TCG → TCT; CAG → CAA; AAG → AAA; GAG → GAA; CGA and CGG → CGC; GGG → GGC.

amplification

The standard PCR protocol was as follows: 50-200 ng genomic DNA was used as a template in the presence of 20-40 μM of each oligos, 400-800 μM dNTP solution, 1 × PCR buffer solution (including 1.5 mM MgCl ₂ ), 2.5 units of TaqI DNA polymerase (using Perkin-Elmer-AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase or Takara Shuzo Taq polymerase).

In some cases PCR was performed by adding 10 μl DMSO or 50 μl 2M betaine optimized.

To one hot Start (addition of polymerase during a preliminary incubation of the entire mixture over 3 minutes at 95 ° C) Each sample underwent a double-step amplification: the first 5 Cycles were carried out by using as the hybridization temperature that of the oligos without the tail of the restriction enzymes was used, followed by 30 cycles consistent with the hybridization temperature of the Oligos in full length carried out were. The cycles were followed by a final extension step over 10 minutes at 72 ° C.

The standard cycles were as follows: denaturation hybridization elongation First 5 cycles 30 seconds, 95 ° C 30 seconds, 50-55 ° C 30-60 seconds, 72 ° C Last 30 cycles 30 seconds, 95 ° C 30 seconds, 65-70 ° C 30-60 seconds, 72 ° C

The Elongation time varied in accordance with the length of the to be amplified ORF.

The Amplifications were all made using either a 9600 or a Perkin-Elmer 2400 GeneAmp PCR system. Around to check the results were Place 1/10 of the volume of amplification on a 1-1.5% agarose gel and the size of each amplified fragment was labeled with a DNA molecular weight marker compared.

The amplified DNA was added either directly to a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume, then onto a 1% agarose gel to be given. The DNA fragment that ties the gang with the right one Size corresponded, was then grown using Qiagen's Gel Extraction Kit Following the manufacturer's instructions elutes and cleansed from the gel. The final volume of the DNA fragment was 30 μl or 50 μl either water or 10 mM Tris, pH 8.5.

Cleavage of PCR fragments

The The purified DNA corresponding to the amplified fragment was analyzed in Divided into 2 aliquots and split twice with: (a) NdeI / XhoI or NheI / XhoI for the cloning into pET-21b + and further expression of the protein as a C-terminus His-tag fusion product; (b) BamHI / XhoI or EcoRI / XhoI for the cloning into pGEX-KG and further expression of the protein as a GST N-terminus fusion product.

For some ORFs can be NheI / BamHI for the cloning is introduced into the vector pTRC-HisA, and others Expression of the protein occurs as an N-terminus His-tag fusion product.

each purified DNA fragment was mixed with 20 units of each restriction enzyme (New England Biolabs) in a final volume of either 30 or 40 μl in the presence the appropriate buffer solution incubated (37 ° C for 3 hours to about Night). The cleavage product was then submerged using the QIAquick PCR purification kit Follow the instructions of the manufacturer and cleaned in one Final volume of 30 (or 50) μl either water or 10mM Tris-HCl, pH 8.5. The final DNA concentration was by 1% agarose gel electrophoresis determined in the presence of a titrated molecular weight label.

Cleavage of the cloning vectors (pET22B, pGEX-KG and pTRC-His A)

10 μg of plasmid was incubated with 50 units of each restriction enzyme in 200 μl of reaction volume In the absence of a suitable buffer solution, incubate overnight at 37 ° C. After applying the entire cleavage product to a 1% agarose gel, the band corresponding to the cleaved vector was purified from the gel using Qiagen QIAquick Gel Extraction Kit and the DNA was dissolved in 50 μl of 10 mM Tris-HCl, pH Value 8.5, eluted. The DNA concentration was determined by measuring the OD _{260 of} the sample and adjusted to 50 μg / μl. 1 μl of the plasmid was used for each cloning procedure.

cloning

The fragments corresponding to the respective ORF, previously cleaved and were purified, ligated into both pET22b and pGEX-KG. In a final volume of 20 μl became a molar ratio of 3: 1 fragment / vector using 0.5 μl of NEB-T4 DNA ligase (400 units / μl) ligated in the presence of the buffer solution supplied by the manufacturer. The reaction mixture was incubated at room temperature for 3 hours. In In some experiments, ligation was performed using the Rapid Ligation Kit "by Boehringer carried out according to the manufacturer.

Around introducing the recombinant plasmid into a suitable strain 100 μl competent E. coli DH5 cells with the ligase reaction solution on ice for 40 minutes, then at 37 ° C for 3 minutes, then after adding 800 μl LB broth again at 37 ° C for 20 minutes incubated. The cells were then in a high speed Centrifuge Eppendorf microfuge and add approximately 200 μl of the supernatant resuspended. The suspension was then switched to LB-ampicillin (100 mg / ml).

The Screening of the recombinant clones was performed by 5 randomly selected Colonies over Night at 37 ° C either in 2 ml (pGEX or pTC clones) or in 5 ml (pET clones) LB broth + 100 μg / ml ampicillin cultured were. The cells were then precipitated and the DNA was submerged Using the QIAprep Spin Miniprep Kit from Qiagen for instructions extracted by the manufacturer to a final volume of 30 ul. 5 μl of each individual miniprp (about 1 g) were cleaved with either NdeI / XhoI or BamHI / XhoI, and the entire cleavage product was along with the molecular weight label (1Kb DNA ladder, GIBCO) on a 1-1.5% agarose gel (depending on of the expected size of the insertion) applied. The screening for the positive clones was done based on the correct insertion size.

cloning

Certain ORFs can be cloned into the vector pGEX-HIS by the EcoRI-PstI cloning sites or EcoRI-SalI or SalI-PstI. After the cloning can the recombinant plasmids are introduced into the E. coli W3110 host cell.

expression

Any ORF that has been cloned into the expression vector can then be transformed into the strain that is suitable for expression of the recombinant protein product. 1 μl of each construct was used to transform 30 μl E. coli BL21 (vector pGEX), E. coli TOP 10 (vector pTRC) or E. coli BL21-DE3 (vector pET) as described above. In the case of the vector pGEX-His, the same E. coli strain (W3110) was used for initial cloning and expression. Individual recombinant colonies were inoculated in 2 ml LB + Amp (100 μg / ml), incubated at 37 ° C overnight, then diluted to 1:30 in 20 ml LB + Amp (100 μg / ml) in 100 ml flasks, ensuring that the OD _{600 was} between 0.1 and 0.15. The flasks were incubated at 30 ° C in rotating water bath swirls until the CD indicated exponential growth suitable for initiation of expression (0.4-0.8 OD for the vectors pET and pTRC; 0.8-1 CD for the vectors pGEX and pGEX-His). For the vectors pET, pTRC and pGEX-His, protein expression was induced by the addition of 1 mM IPTG, while the final concentration of IPTG was 0.2 mM in the case of the pGEX system. After incubation for 3 hours at 30 ° C, the final concentration of the sample was checked by the CD. To check expression, 1 ml was removed from each sample, centrifuged in a microfuge, the precipitate resuspended in PBS and analyzed by 12% SDS-PAGE with Coomassie blue staining. The entire sample was centrifuged at 6000 g and the precipitate resuspended in PBS for further use.

Purification of GST fusion proteins in big scale

A single colony was grown overnight at 37 ° C in an LB + amp agar dish. The Bak Tetras were inoculated in 20 ml of LB + Amp liquid culture in a water bath and cultured overnight. The bacteria were diluted 1:30 in 600 ml of fresh culture medium and allowed to grow at an optimum temperature (20-37 ° C) to an OD ₅₅₀ of 0.8-1. Protein expression was induced with 0.2 mM IPTG followed by incubation for three hours. The culture was centrifuged at 8000 rpm at 4 ° C. The supernatant was discarded and the bacterial pellet resuspended in 7.5 ml of cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40 W using a B-15 Brans ultrasound machine, twice frozen and thawed, and centrifuged again. The supernatant was collected and mixed with 150 μl glutathione Sepharose 4B resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was washed twice with 10 ml of cold PBS for 10 minutes, resuspended in 1 ml of cold PBS and applied to a commercial column. The resin was washed twice with 2 ml of cold PBS until the flow had reached an OD ₂₈₀ of 0.02-0.06. The GST fusion protein was eluted by the addition of 700 μl cold glutathione elution buffer solution (10 mM reduced glutathione, 50 mM Tris-HCl) and the fractions were collected until the OD _{280 was} 0.1. 21 μl of each fraction was loaded on a 12% SDS gel using either the molecular weight standard for the broad area Biorad SDS-PAGE study (M1) (200, 116, 25, 97, 4, 66, 2). 45, 31, 21.5, 14.4, 6.5 kDa) or Amersham's Rainbow marker (M ") (220, 66, 46, 30, 21.5, 14.3 kDa) were used as standards were. Since the MG of GST is 26 kDa, this value must be added to the MG of each GST fusion protein.

Purification of soluble His fusion proteins in large scale

A single colony was cultured overnight at 37 ° C in an LB + amp agar dish. The bacteria were seeded in 20 ml of LB + Amp liquid culture and incubated overnight in a water bath swivel. The bacteria were diluted 1:30 in 600 ml of fresh culture medium and allowed to grow at an optimum temperature (20-37 ° C) to an OD ₅₅₀ of 0.6-0.8. Protein expression was induced by the addition of 1 mM IPTG and the culture was further incubated for three hours. The culture was centrifuged at 8000 rpm at 4 ° C, the supernatant was discarded, and the bacterial pellet was dissolved in 7.5 ml cold 10 mM imidazole buffer solution (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8 ) resuspended. The cells were disrupted by ultrasonication on ice for 30 sec at 40 W using a Branson B-15 ultrasound machine, twice frozen and thawed, and centrifuged again. The supernatant was collected and mixed with 150 μl of Ni ²⁺ resin (Pharmacia) (previously washed with 10 mM imidazole buffer solution) and incubated at room temperature with gentle stirring for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was washed twice with 10 ml cold 10 mM imidazole buffer solution for 10 minutes, resuspended in 1 ml cold 10 mM imidazole buffer solution and applied to a commercial column. The resin was washed at 4 ° C with 2 ml cold 10 mM imidazole buffer solution until the flow-through solution reached OD ₂₈₀ 0.02-0.06. The resin was washed with 2 ml of cold 20 mM imidazole buffer solution (300 mM NaCl, 50 mM phosphate buffer solution, 20 mM imidazole, pH 8) until the flow-through solution reached OD ₂₈₀ 0.02-0.06 , The His fusion protein was eluted by the addition of 700 μl cold 250 mM imidazole buffer solution (300 mM NaCl, 50 mM phosphate buffer solution, 250 mM imidazole, pH 8) and the fractions were collected until the OD was ₂₈₀ , 1 was. 21 μl of each fraction was applied to a 12% SDS gel.

Purification of insoluble His fusion proteins in large scale

A single colony was cultured overnight at 37 ° C in an LB + amp agar dish. The bacteria were inoculated into 20 ml of a LB + Amp liquid culture in a water bath pan and cultured overnight. The bacteria were diluted 1:30 in 600 ml of fresh culture medium and allowed to grow at optimum temperature (37 ° C) to OD ₅₅₀ of 0.6-0.8. Protein expression was induced by the addition of 1 mM IPTG and the culture was further incubated for three hours. The culture was centrifuged at 8000 rpm at 4 ° C. The supernatant was discarded and the bacterial pellet resuspended in 7.5 ml of Buffer B (urea 8M, 10mM Tris-HCl, 100mM phosphate buffer, pH 8.8). The cells were disrupted by ultrasonication on ice for 30 sec at 40 W using a Branson B-15 ultrasound machine, twice frozen and thawed, and centrifuged again. The supernatant was stored at -20 ° C while the precipitates were resuspended in 2 ml guanidine buffer solution (6 M guanidine hydrochloride, 100 mM phosphate buffer solution, 10 mM Tris-HCl, pH 7.5) and incubated for 10 cycles a homogenizer were treated. The product was centrifuged at 13,000 rpm for 40 minutes. The supernatant was mixed with 150 μl of Ni ²⁺ resin (Pharmacia) (previously washed with buffer solution B) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was transferred twice with 10 ml Buffer B Washed for 10 minutes, resuspended in 1 ml Buffer B and applied to a disposable column. The resin was washed at room temperature with 2 ml of Buffer B solution until the flow through solution reached OD ₂₈₀ of 0.02-0.06. The resin was washed with 2 ml buffer solution C (urea 8 M, 10 mM Tris-HCl, 100 mM phosphate buffer solution, pH 6.3) until the flow-through solution reached OD ₂₈₀ 0.02-0.06. The His fusion protein was eluted by the addition of 700 μl of elution buffer solution (urea 8 M, 10 mM Tris-HCl, 100 mM phosphate buffer solution, pH 4.5) and the fractions were collected until the OD _{280 was} 0.1 , 21 μl of each fraction was applied to a 12% SDS gel.

Renaturation of His fusion proteins

10% glycerol was added to the denatured proteins. The proteins were then reduced to 20 μg using dialysis buffer solution I (10% glycerol, 0.5 M arginine, 50 mM phosphate buffer solution, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 M urea, pH 8.8) / ml and dialysed against the same buffer solution at 4 ° C for 12-14 hours. The protein was further challenged with dialysis buffer solution II (10% glycerol, 0.5 M arginine, 50 mM phosphate buffer solution, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, pH 8.8) for 12-14 hours at 4 ° C dialysed. The protein concentration was calculated according to the following formula: Protein (mg / ml) = (1.55 x OD 280 ) - (0.76 x OD 260 )

Immunizations of mice

20 μg of each purified protein is used to immunize mice intraperitoneally. In the case of some ORFs, Balb C mice were immunized with Al (OH) ₃ as adjuvant on days 1, 21 and 42, and the immune response was observed in samples taken on day 56. For other ORFs, CD1 mice could be immunized according to the same protocol. For ORFs 25 and 40, CD1 mice were immunized using Freund's adjuvant, and the same immunization protocol was used with the change that the immune response was measured at day 42 instead of 56. Similarly, for still other ORFs, CD1 mice were immunized with Freund's adjuvant, but the immune response was measured on day 49.

ELISA test (serum analyzes)

The unencapsulated strain MenB M7 was plated on chocolate agar plates and incubated overnight at 37 ° C. Bacterial colonies were collected from the agar plates with a sterile Dracon swab and inoculated into 7 ml of Mueller-Hinton broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by monitoring the OD ₆₂₀ . The bacteria were allowed to grow until the OD reached 0.3-0.4. The culture was centrifuged for 10 minutes at 10,000 rpm. The supernatant was discarded and the bacteria were washed once with PBS, resuspended in PBS containing 0.025% formaldehyde and incubated for 2 hours at room temperature and then overnight at 4 ° C with stirring. 100 μl of bacterial cells were added to each well of a 96-well Greiner plate and incubated overnight at 4 ° C. The wells were then washed three times with PBT wash buffer solution (0.1% Tween-20 in PBS). 200 μl of saturation buffer solution (2.7% polyvinylpyrrolidone 10 in water) was added to each well and the plates were incubated for 2 hours at 37 ° C. The wells were washed three times with PBT. 200 μl of diluted sera (dilution buffer solution: 1% BSA, 0.1% Tween-20, 0.1% NaN ₃ in PBS) were added to each well and the plates were incubated for 90 minutes at 37 ° C. The wells were washed three times with PBT. 100 μl rabbit HRP-conjugated anti-mouse (Dako) serum diluted 1: 2000 in dilution buffer solution was added to each well and the plates were incubated for 90 minutes at 37 ° C. The wells were washed three times with PBT buffer solution. 100 μl of substrate buffer solution for HRP (25 ml citrate buffer solution, pH 5, 10 mg O-phenyldiamine and 10 μl H ₂ O) were added to each well and the plates were allowed to stand at room temperature for 20 minutes. 100 μl H ₂ SO _{4 was} added to each well and the OD ₄₉₀ was tracked. The ELISA test was considered positive when the OD _{490 was} 2.5 times the corresponding preimmune sera.

Method of FACScan Bacterial Binding Assay

The unencapsulated strain MenB M7 was applied to chocolate agar plates and incubated overnight at 37 ° C. Bacterial colonies were collected from the agar plates with a sterile Dracon swab and placed in four tubes each containing 8 ml of Mueller-Hinton broth (Difco) containing 0.25% glucose, inoculated. Bacterial growth was monitored every 30 minutes by monitoring the OD ₆₂₀ . The bacteria were allowed to grow until the OD reached 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the precipitate was resuspended in blocking buffer solution (1% BSA, 0.4% NaN ₃ ) and centrifuged for 5 minutes at 4000 rpm. The cells were resuspended in blocking buffer solution to reach an OD ₆₂₀ of 0.07. 100 μl of bacterial cells were added to each well of a 96-well Costar plate. 100 μl of diluted (1: 200) sera (in blocking buffer solution) were added to each well and the plates were incubated for 2 hours at 4 ° C. The cells were centrifuged for 5 minutes at 4000 rpm, the supernatant was aspirated, and the cells were washed by the addition of 200 μl of blocking buffer solution / well to each well. 100 μl of goat R-phycoerythrin-conjugated anti-mouse F (ab) ₂ diluted 1: 100 was added to each well and the plates were incubated for 1 hour at 4 ° C. The cells were seeded by centrifugation at 4000 rpm for 5 minutes and washed by adding 200 μl of blocking buffer solution / well. The supernatant was aspirated and the cells were resuspended in 200 μl of PBS, 0.25% formaldehyde / well. The samples were transferred to FACScan tubes and measured. The conditions for the FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H threshold: 92; FSC-PMT voltage: E 02; SSC-PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539. Compensation values: 0.

OMV preparations

bacteria were over Night bred on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20 mM Tris-HCl. The heat inactivation was carried out at 56 ° C for 30 minutes, and the bacteria were disrupted by ultrasound over 10 'on ice (50% duty cycle, 50% power). Unbroken cells were removed by centrifugation with 5000 g over 10 minutes away, and the entire fraction of cell envelopes was recovered by centrifugation with 50,000 g at 4 ° C over 75 minutes. Around to extract cytoplasmic membrane proteins from the outer raw membranes, the entire fraction was resuspended in 2% sarcosyl (Sigma) and at room temperature over Incubated for 20 minutes. The suspension was taken at 10,000 g for 10 minutes centrifuged to remove aggregates, and the supernatant was about it out with 50,000 g over Ultracentrifuged for 75 minutes to precipitate the outer membranes. The outer membranes were resuspended in 10 mM Tris-HCl, pH 8, and the protein concentration was tested by the Bio-Rad protein assay using BSA as a standard measured.

Preparation of the total extracts

bacteria were over Night bred on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was carried out at 56 ° C for 30 minutes.

Western blot method

purified Proteins (500 ng / lane), outer membrane vesicles (5 μg) and Total cell extracts (25 μg), derived from MenB strain 2996 were subjected to 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was over 2 hours with 150 mA at 4 ° C in transfer buffer solution (0.3% Tris base, 1.44% glycine, 20% methanol). The membrane was by overnight incubation at 4 ° C in saturation buffer solution (10% Skimmed milk, 0.1% Triton X100 in PBS). The membrane was twice with washing buffer solution (3% skimmed milk, 0.1% Triton X100 in PBS) and over 2 hours at 37 ° C with mouse sera diluted 1: 200 in washing buffer solution. The membrane was washed twice and over 90 minutes with horseradish peroxidase labeled 1: 2000 ratio diluted incubated with anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti 4CN substrate kit (Bio-Rad). The reaction was stopped by adding water.

Examination for germ killing

Strain MC5B was grown overnight at 37 ° C on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7 ml of Mueller Hinton broth. The suspension was incubated at 37 ° C on a nutator and allowed to grow until the OD _{620 was} between 0.5-0.8. The culture was aliquoted into sterile 1.5 ml Eppendorf tubes and centrifuged for 20 minutes at full speed in a microfuge. The precipitate was washed once in Gey buffer solution (Gibco) and resuspended in the same buffer solution to an OD ₆₂₀ of 0.5, diluted 1: 20,000 in Gey buffer and stored at 25 ° C.

50 μl of Gey buffer solution / 1% BSA were added to each well of a 96-well tissue culture plate added. 25 μl diluted (1: 100) mouse sera (dilution buffer solution: Gey buffer solution / 0.2% BSA) were added to each well and the plate was incubated at 4 ° C. 25 μl of The bacterial suspension described above was added to each well added. 25 μl from either heat-inactivated (56 ° C water bath over 30 minutes) or normal Baby rabbit complement was added to each well. Immediately after the addition of the baby rabbit complement were 22 μl of each sample / well plated on Mueller Hinton agar plates (Time 0). The 96-well plate was over 1 hour at 37 ° C incubated with rotation, and then 22 μl of each sample / well plated on Mueller-Hinton agar plates (time 1). To Incubation over Night, the colonies were the time 0 and time 1 h corresponded, counted.

sequences

Using the methods described above, the following section of the DNA sequence of N. gonorrhoeae has been identified <SEQ ID NO: 1>:

Der nachstehende Abschnitt einer DNA-Teilsequenz wurde in N. meningitidis identifiziert <SEQ ID NO: 3>:

This corresponds to the amino acid sequence <SEQ ID NO: 2; ORF 741.ng>:

The following section of a partial DNA sequence was identified in N. meningitidis <SEQ ID NO: 3>:

This corresponds to the amino acid sequence <SEQ ID NO: 4; ORF 741>:

The following section of the DNA sequence was identified in N. meningitidis <SEQ ID NO: 5>:

This corresponds to the amino acid sequence <SEQ ID NO: 6; ORF 741.a>:

SEQUENCE LISTING

Claims (40)

Protein comprising: The amino acid sequence SEQ ID NO: 2; An amino acid sequence, which is a 50% or higher sequence identity with SEQ ID NO: 2 and which is effective for preventing a Disease caused by Neisseria bacteria; An amino acid sequence, comprising a fragment of 8 or more contiguous amino acids SEQ ID NO: 2, wherein the fragment comprises an epitope of SEQ ID NO: 2; The amino acid sequence SEQ ID NO: 4; • one Amino acid sequence which is a 50% or higher sequence identity with SEQ ID NO: 4 and which is effective for preventing a Disease caused by Neisseria bacteria; An amino acid sequence, comprising a fragment of 10 or more contiguous Amino acids SEQ ID NO: 4, wherein the fragment comprises an epitope of SEQ ID NO: 4; The amino acid sequence SEQ ID NO: 6; • one Amino acid sequence which is a 50% or higher sequence identity with SEQ ID NO: 6 and which is effective for preventing a Disease caused by Neisseria bacteria; An amino acid sequence, comprising a fragment of 10 or more contiguous Amino acids SEQ ID NO: 6, wherein the fragment comprises an epitope of SEQ ID NO: 6. The protein of claim 1 which has sequence identity with SEQ ID NO: 4, the higher than 60%. A protein according to claim 2, which has a sequence identity with SEQ ID NO: 4, the higher than 70% is. A protein according to claim 3, which has a sequence identity with SEQ ID NO: 4, the higher than 80% is. A protein according to claim 4, which has a sequence identity with SEQ ID NO: 4, the higher than 90% is. A protein according to claim 5, which has a sequence identity with SEQ ID NO: 4, the higher than 95% is. A protein according to claim 6, which has a sequence identity with SEQ ID NO: 4, the higher than 99% is. A protein according to claim 1, comprising an amino acid sequence, which is a fragment of 12 or more contiguous amino acids SEQ ID NO: 4. A protein according to claim 8, comprising an amino acid sequence, which is a fragment of 14 or more contiguous amino acids SEQ ID NO: 4. A protein according to claim 9, comprising an amino acid sequence, which is a fragment of 16 or more contiguous amino acids SEQ ID NO: 4. A protein according to claim 10, comprising an amino acid sequence, which is a fragment of 18 or more contiguous amino acids SEQ ID NO: 4. A protein according to claim 11, comprising an amino acid sequence, which is a fragment of 20 or more contiguous amino acids SEQ ID NO: 4. A protein according to claim 1, comprising an amino acid sequence, which is a fragment of 10 or more contiguous amino acids SEQ ID NO: 2 comprises. A protein according to claim 13, comprising an amino acid sequence, which is a fragment of 12 or more contiguous amino acids SEQ ID NO: 2 comprises. A protein according to claim 14, comprising an amino acid sequence, which is a fragment of 14 or more contiguous amino acids SEQ ID NO: 2 comprises. A protein according to claim 15, comprising an amino acid sequence, which is a fragment of 16 or more contiguous amino acids SEQ ID NO: 2 comprises. A protein according to claim 16, comprising an amino acid sequence, which is a fragment of 18 or more contiguous amino acids SEQ ID NO: 2 comprises. A protein according to claim 17, comprising an amino acid sequence, which is a fragment of 20 or more contiguous amino acids SEQ ID NO: 2 comprises. A protein according to any one of the preceding claims, wherein the protein is a Neisseria antigen is. Antibody, which specifically binds to an amino acid sequence that is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. antibody according to claim 20, which is a monoclonal antibody. Nucleic acid molecule, which a protein according to any one of claims 1 to 19 coded. Nucleic acid molecule according to claim 22, comprising a nucleotide sequence selected from the group consisting from SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. Nucleic acid molecule, which encodes a protein that is effective for the prevention of diseases due to Neisseria bacteria, comprising (i) a fragment of 12 or more contiguous Nucleotides of SEQ ID NO: 1, which is an epitope of SEQ ID NO: 2 encode; (ii) a fragment of 20 or more contiguous Nucleotides of SEQ ID NO: 3, which is an epitope of SEQ ID NO: 4 encode; or (iii) a fragment of 20 or more contiguous Nucleotides of SEQ ID NO: 5, which is an epitope of SEQ ID NO: 6 encode. Nucleic acid molecule according to claim 24, comprising a fragment of 25 or more contiguous Nucleotides from SEQ ID NO: 3. Nucleic acid molecule according to claim 25, comprising a fragment of 30 or more contiguous Nucleotides from SEQ ID NO: 3. Nucleic acid molecule according to claim 26, comprising a fragment of 35 or more contiguous Nucleotides from SEQ ID NO: 3. Nucleic acid molecule according to claim 27, comprising a fragment of 40 or more contiguous Nucleotides from SEQ ID NO: 3. A nucleic acid molecule comprising a nucleotide sequence which is complementary to a nucleic acid molecule after a the claims 22 to 28 is. A composition comprising a protein, a nucleic acid molecule or a antibody according to one of the preceding claims. A composition according to claim 30 which is an immunogenic Composition is. A composition according to claim 30 which is a vaccine composition or a diagnostic composition. A composition according to claim 30 for use as a medicine. Use of a protein according to any one of claims 1 to 19, a nucleic acid molecule one of the claims 22 to 29 or an antibody according to claim 20 or 21 for the preparation of a medicament for the prevention of infection, which is caused by Neisseria bacteria. Use according to claim 34, wherein the medicament for the prevention of an infection caused by N. meningitidis is, serves. Use according to claim 35, wherein the medicament for the prevention of an infection caused by N. meningitidis B. is, serves. A composition according to any one of claims 31 to 33 or use according to any one of claims 34 to 36, wherein the composition or the drug contains an adjuvant. A vector comprising the nucleotide sequence of a nucleic acid molecule one of the claims 22 to 29. A host cell transformed with the vector of claim 38th Process for producing a protein according to a the claims 1-19, comprising the step of growing a host cell Claim 39 under conditions which induce protein expression.